Author Topic: Enzyme Sequences (Read 5944 times)

Xenonpill · « **on:** February 11, 2001, 08:00:00 PM »

These are from The Resonance Project v2.15 winter 97/98.
Some of the article is available on the resonance project's website.

Tryptophan Hydroxylase:
Amino Acids: F,S,Q,E,I,G,L,A
Nucleuotides (human, rat, rabbit, mouse): TTC,TCC,CAA,GAA,ATT,GGC,CTG,GCT

Tryptophan Decarboxylase:
Amino Acids: G,K,E,M,V,D,Y
Nucleotides:
Human: GGG,AAG,GAG,ATG,GTG,GAT,TAC
Cow: GGG,AAG,GAG,ATG,GTG,GAT,TAC
Rat: GGG,AAG,GAG,ATG,GTG,GAT,TAT

Hydroxyindole O-Methyltransferase:
Amino Acids: D,A,V,R,E,G,R,N
Nucleotides:
Human: GAC,GCC,GTG,AGA,GAA,GGA,AGG,AAC
Chicken: GAT,GCT,GTG,AGA,GAA,GGA,AGA,AAT

Unfortunatly, no references were supplied in the print article or at www.resproject.com, however, it may be possible to get the refs by bugging someone at the resonance project.
KrZ, would this kind of thing help you make a yeast that produces gobs of DMT when mixed with water?

Xenonpill

KrZ · « **Reply #1 on:** February 12, 2001, 07:59:00 AM »

Tryptophan->Tryptamine
DEFINITION C.roseus tdc gene for tryptophan decarboxylase.
ACCESSION   X67662
/translation="MGSIDSTNVAMSNSPVGEFKPLEAEEFRKQAHRMVDFIADYYKN    VETYPVLSEVEPGYLRKRIPETAPYLPEPLDDIMKDIQKDIIPGMTNWMSPNFYAFFP ATVSSAAFLGEMLSTALNSVGFTWVSSPAATELEMIVMDWLAQILKLPKSFMFSGTGG GVIQNTTSESILCTIIAARERALEKLGPDSIGKLVCYGSDQTHTMFPKTCKLAGIYPN NIRLIPTTVETDFGISPQVLRKMVEDDVAAGYVPLFLCATLGTTSTTATDPVDSLSEI    ANEFGIWIHVDAAYAGSACICPEFRHYLDGIERVDSLSLSPHKWLLAYLDCTCLWVKQ    PHLLLRALTTNPEYLKNKQSDLDKVVDFKNWQIATGRKFRSLKLWLILRSYGVVNLQS HIRSDVAMGKMFEEWVRSDSRFEIVVPRNFSLVCFRLKPDVSSLHVEEVNKKLLDMLN    STGRVYMTHTIVGGIYMLRLAVGSSLTEEHHVRRVWDLIQKLTDDLLKEA"

DEFINITION Oryctolagus cuniculus indolethylamine N-methyltransferase (INMT) mRNA, complete cds.
ACCESSION   AF077826
/translation="MEGGFTGGDEYQKHFLPRDYLNTYYSFQSGPSPEAEMLKFNLEC LHKTFGPGGLQGDTLIDIGSGPTIYQVLAACESFKDITLSDFTDRNREELAKWLKKEP    GAYDWTPALKFACELEGNSGRWQEKAEKLRATVKRVLKCDANLSNPLTPVVLPPADCV LTLLAMECACCSLDAYRAALRNLASLLKPGGHLVTTVTLQLSSYMVGEREFSCVALEK    EEVEQAVLDAGFDIEQLLYSPQSYSASTAPNRGVCFLVARKKPGS"

This can also catalyze the second methylation according to quicksilver (who has been alot of help, and I am very appreciative of).

Complete mRNAs for every step, nothing too gigantic either...

Quicksilver · « **Reply #2 on:** February 12, 2001, 11:22:00 PM »

KrZ-
Go ahead and post my last PM if you still have it. Maybe there are enough bees with molec' bio' background to find that last step that I couldn't.

Basically my work ended in an e.coli that produces DMT (not psilocin) because there is no cloned enzyme that will hydroxylate the 4 position on DMT. (and thus convert DMT->psilocin) Not a total loss, but not what we were looking for.

Xenopill-
If you know of a indole-hydroxylase, or tryptamine hydroxylase that will work, you'll make me a happy bee.

KrZ, thanks for the acknowledgement. In doing so, it reaffirms what most of us know about your strength of character already.

-quicksilver-

Rhodium · « **Reply #3 on:** February 13, 2001, 05:40:00 AM »

Quicksilver: Are yuo telling me that there already exist a bacterium which can convert tryptophan to DMT? How many millimoles can a culture churn out per hour?

http://rhodium.lycaeum.org

KrZ · « **Reply #4 on:** February 13, 2001, 11:10:00 AM »

KrZ-
I wasn't able to get any of the articles, but I'm not sure of their worth anyway. (since they all pre-date molecular bio/DNA techniques)

Ironically, the textbook Biochemistry by Mathews/vanHolde Chapter 21 page 740, review question 4, actually asks for a biosynthesis of
psilocybin from tryptophan. Too bad there's no answers in the book.....

Regardless, you stated:
Tryptophan -1-> Tryptamine -2-> N-Methyl-tryptamine -3-> N,N-dimethyltryptamine -4-> psilocin -5-> psilocybin

The enzymes for the first 3 steps are known, and the protein+DNA sequences are known too.
1-Tryptophan->Tryptamine
   Enzyme=aromatic amino acid decarboxylase
   AKA: AAAD, AKA: dopadecarboxylase
   Genbank Accession# M88700 (protein sequence=AAA20894)
   Reference:Human dopa decarboxylase: localization to human chromosome 7p11 and characterization of hepatic cDNAs. Genomics 13
(2), 469-471 (1992)

2-Tryptamine -> N-Methyl-tryptamine
   Enzyme=Indolethylamine-N-Methyltransferase
   AKA INMT
   Genebank Accession# NM_006774
   Reference:Human indolethylamine N-methyltransferase: cDNA cloning and expression, gene cloning, and chromosomal localization.
Genomics 61 (3), 285-297 (1999)

3-N-Methyl-tryptamine -> N,N-DiMethyl-tryptamine
   Same as #2

4-N,N-dimethyltryptamine -> psilocin
   There most likely isn't a human gene for this. (otherwise we'd all be alot happier people)
   I find no references in medline for: indole-hydroxylase, trptamine-hydoxylase, or indolamine-hydroxylase, leading me to believe
there is no human (or other species) homolog. Dead end? My first thoughts are maybe the psilocin step is impossible at this time, or
at least until someone clones the gene responsible. So where from here? Perhaps the 5-hydroxyderivative would be more attainable.
See 5-OH-N,N-DMT

http://www.erowid.org/library/books_online/tihkal/tihkal19.shtml

Shulgin is less than enthusiastic about this compound.
This could be accomplished with tryptophan hydroxylase. Worth the effort?? I'm skeptical.

5-psilocin -> psilocybin
Haven't looked yet.

That's all my ramblings. Any feedback would be much appreciated.
Cheers.
-quicksilver-

In reply to:

1. Agurell, S., Blomkvist,S. and Catalfomo, P.. "Biosynthesis of Psilocybin in Submerged Culture of Psilocybin cubensis: Part I.
Incorporation of labeled tryptophan and tryptamine," Acta Pharm. Suecica, Vol. 3 (1966), 37-44.

2. Agurell, S., Lars, J. and and Nilsson, C.. "Biosynthesis of Psilocybin: Part II. Incorporation of Labelled Tryptamine Derivatives, Acta
Chemica Scandinavica, Vol. 22, No. 4 (1968), 1210-1218.

6. Catalfomo, P. and Tyler, V. E., Jr.. "The Production of Psilocybin in Submerged Culture by Psilocybe cubensis, Lloydia, Vol. 27, No.l
(March 11. Leung, A.Y. and Paul, A.G.. "The Relationship of Carbon and Nitrogen Nutrition of Psilocybe baecocystis to the Production of
Psilocybin and its Analogs," Lloydia, Vol. 32, No. 1(March, 1969), 66-71.1964), 53-63.

Lilienthal · « **Reply #5 on:** February 13, 2001, 11:57:00 AM »

Here's an interesting link for you:

http://www.fortunecity.com/meltingpot/gregory/1042/

. It contains a huge list of literature!

KrZ · « **Reply #6 on:** February 13, 2001, 02:18:00 PM »

The INMTs are heavily inhibited by DMT, I'm not so sure they are going to be ideal for process fermentation. Still you could isolate the eukaryotic mRNAs, use RT conversion to make the DNA sequence (no introns), PCR, ecUP-Plasmid insert it, et voila. However, they don't use recombinant e. coli to make aspergillus antibiotics, and there is alot of information about mycofermentation and strain optimization for fungii... Need to grab the Agurell articles...

Quicksilver · « **Reply #7 on:** February 13, 2001, 06:10:00 PM »

Rhodium-

Quicksilver: Are yuo telling me that there already exist a bacterium which can convert tryptophan to DMT? How many millimoles can a culture churn out per hour?

No, but hypothetically one could 'easily' make one. I was/am interested in a bacteria that could make psilocin, but I don't think there is a cloned cDNA for a 4'-indole-hydroxylase. I've found a cytochome p450 (CYP2D6) that will 4'-hydroxylate debrisoquine. (but debrisoquine is not quite an indole, mind you it is similar) And of course there are enzymes that 5'-hydroxylate indoles (such as tryptophan). S. Agurell does seem to be one of the most knowledgable/early forefathers in the field of psilocin biosynthesis.

-quicksilver-

Quicksilver · « **Reply #8 on:** February 13, 2001, 06:16:00 PM »

Lilienthal-
How did you find this link?
Could you post the contents of that word file please.
Thanks,
-quicksilver-

Lilienthal · « **Reply #9 on:** February 14, 2001, 03:02:00 AM »

I searched Altavista for '+psilocybin +cloning'. The wordfile seems to be much too big to be posted here.

bioboy · « **Reply #10 on:** February 14, 2001, 03:36:00 AM »

1: Plant Mol Biol 1997 Aug;34(6):935-48


   Molecular cloning and characterization of desacetoxyvindoline-4-hydroxylase, a
   2-oxoglutarate dependent-dioxygenase involved in the biosynthesis of vindoline in
   Catharanthus roseus (L.) G. Don.

A 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.11) which catalyzes the 4-hydroxylation of
desacetoxyvindoline was purified to homogeneity. Three oligopeptides isolated from a tryptic digest of the
purified protein were microsequenced and one oligopeptide showed significant homology to hyoscyamine 6
beta-hydroxylase from Hyoscyamus niger. A 36-mer degenerate oligonucleotide based on this peptide sequence was used to screen a Catharanthus roseus cDNA library and three clones, cD4H-1 to -3, were isolated. Although none of the three clones were full-length, the open reading frame on each clone encoded a putative protein containing the sequence of all three peptides. Primer extension analysis suggested that cD4H-3, the longest cDNA clone, was missing 156 bp at the 5' end of the clone and sequencing of the genomic clone,
gD4H-8, confirmed these results. Southern blot analysis suggested that d4h is present as a single-copy gene in C. roseus which is a diploid plant, and the significant differences in the sequence of the 3'-UTR between
cD4H-1 and -3 suggest that they represent dimorphic alleles of the same hydroxylase. The identity of the clone
was further confirmed when extracts of transformed Escherichia coli expressed D4H enzyme activity. The D4H
clone encoded a putative protein of 401 amino acids with a calculated molecular mass of 45.5 kDa and the
amino acid sequence showed a high degree of similarity with those of a growing family of
2-oxoglutarate-dependent dioxygenases of plant and fungal origin.

KrZ · « **Reply #11 on:** February 14, 2001, 08:18:00 AM »

I pulled out some of the best parts for you quicksilver, neat stuff;

Psilocybin biosynthesis
4-Hydroxylated or 4-methoxylated indoles are very rare in nature. The only known examples beside the psilocybin-type alkaloids are the 4-hydroxylation of indole acetic acid by Aspergillus niger strains (54), methoxylated b-carbolines from Banisteriopsis argentea and Picrasma javanica, the reserpine analog venenatine from Alstonia venenata, the yohimbine analog mitragynine from Mitragyna speciosa (79, 82 p. 703), and the aminopyrimidyl-indolic meridianines from the tunicate Aplidium meridianum (25). Psilocybin has a simple structure in contrast to the former alkaloids. It is formally derived from tryptophan in five distinct biosynthetic reactions, i.e. decarboxylation, indole-hydroxylation, two N-methylations, and O-phosphorylation (Table 1). Feeding experiments with putative intermediates, analogs of them, or radioactive precursors supported the view that tryptophan decarboxylation is the first biosynthetic step and that O-phosphorylation is the final step. The sequence of the remaining intermediate reaction steps is still unclear. Some authors even suppose a biosynthetic grid with multiple routes to psilocybin (Figure 1) (13, 5, 78).

Abbreviation   Full name
Decarboxylase   Tryptophan Decarboxylase
Hydroxylase   Tryptamine 4-Monooxygenase,
Tryptamine :    Oxygen Oxidoreductase, 4-hydroxylating
Methylase      Tryptamine-w-amino-methyltransferase
Phosphorylase   Psilocin-O-phosphotransferase

Table 1. The psilocybin synthesizing enzymes. The full name follows standard enzyme nomenclature while the abbreviated name is used throughout this text. The enzymes may actually consist of several related enzyme with slightly different substrate specifities.

Expression cloning procedure
For a definitive evaluation of psilocybin biosynthesis, the participating enzymes need to be isolated and tested for their substrate specifities and activities. The feasible way to do this was by genetically cloning and heterologous expression.
The cloning methodology chosen is an alternative to PCR based procedures and has been devoloped and used succesfully to clone a broad range of fungal enzymes (21, 20, 15, 14, 53). It consists of ligating a cDNA library into an expression vector (pYes2), transforming a host organism (S. cerevisiae), and a screening the resulting colonies for enzyme activity (Figure 2).
The success of such expression cloning procedures depends on reliable and sensitive enzyme assays for the colony screening step. Indeed it was possible to find such assays for all enzymes of the psilocybin biosynthesis pathway (Table 2).
A second important requirement is the use of biomaterial containing high amounts of the respective enzymes mRNA. Usually this is a growing tissue containing the metabolites of interest. In this study still developing Psilocybe tampanensis sclerotia were used for mRNA extraction. They grew rapidly and reliably on a special medium and contained high amounts of psilocybin and psilocin.

Total RNA isolation from enzyme producing biomaterial
            \/
         mRNA preparation
            \/
         cDNA synthesis
            \/
Ligation into E. coli / S. cerevisiae shuttle expression vector (pYes2)
            \/
      Amplification in E. coli
            \/
Colony pooling and plasmid preparation (20 pools of 5,000 colonies each)
            \/
      Yeast transformations
            \/\/\/\/
Enzyme activity screenings of colonies by color reactions
after inducing expression (galactose)
            \/
      Rescreening positive clones
            \/
Cross-transformation of E. coli, insert sequencing and analysis

Figure 2. Expression cloning flow scheme. As used by Dalbøge et al. to clone a broad range of fungal enzymes.

Enzyme      Screening procedure
Decarboxylase   5-fluoro-tryptophan resistance
Hydroxylase   feeding tryptamines, Keller's reaction
Methylase   feeding tryptamines, derivatization and removal of substrate, radioactive detection
Phosphorylase   feeding psilocin, derivatization of substrate, Keller's reaction after phosphatase treatment

Table 2. Enzyme activity screening procedures. All tests can be performed on colonies to allow a parallel screening procedures. The Keller's reaction is a very sensitive and specific color reaction for hydroxylated indoles.

Mushroom media and culturing
Media ingredients and sources: dried unrefined sugar-cane extract (Rapadura, organic food or third world stores), sugar-beet syrup (75% dry matter, food stores), mixed pollen (organic or health food stores), dry yeast extract (Oxoid, England), peptone (Difco Laboratories), agar (Merck, Germany), commercial malt extract / yeast solutions "Salvator" (containing 18.3% stammwuerze) and "Hefe Weissbier dunkel" (dark unfiltered wheat beer, containing 12.4% stammwuerze, both from Paulaner, Munich, Germany).
The mycelia were cultured on Parafilm sealed MEY plates (6% malt extract syrup, 0.6% yeast extract, 1.5% agar) at 28°C ± 2°C in an incubator (6). For propagation 1 cm x 1 cm blocks were cut out and transferred to the middle of a new agar plate under sterile conditions.
Plant hormones were added as ethanolic solutions. Media containing KH2PO4, pollen, acetic acid, citric acid, and ascorbic acid medium were autoclaved after adjusting the pH. Media from beer (pH around 5.5) were autoclaved for 40 min.

Mushroom extraction
For a simple but efficient extraction of psilocin as well as psilocybin the following method was applied (98, 58, 45). 7.5 mg lyophilized mushroom material was homogenized in a 1 ml glass mortar with 250 µl methanolic extraction solvent (75% MeOH, 0.1% ascorbic acid). This suspension and 2 x 125 µl methanolic rinsings were pooled in a microcentrifuge tube. After agitation for 10 min at RT the tube was centrifuged for 2 min at 14,000 rpm at RT (5415C centrifuge), the supernatant was transferred to a fresh tube, and the pellet was resuspended in 250 µl ethanolic extraction solvent (75% EtOH, 0.1% ascorbic acid). After agitation for 10 min at RT and centrifugation as above the supernatants were pooled and stored at -20°C in an airtight microcentrifuge tube.

Reagents and procedures from the cDNA Synthesis System Kit (Gibco BRL) were used in combination with a vector primed cDNA synthesis literature method (85). This approach was not succesful.

Here's a cool transformant screening procedure;
5-Fluoro-tryptophan decarboxylase screening
For tryptophan decarboxylase screening transformed FY 73 cells were plated directly onto SC-gal containing 0.5 - 1.5 mM 5-fluoro-tryptophan and were incubated up to 6 days at 30°C.

No-Go for submerged culture;
The tested Psilocybe strains showed variable preferences for the tested media. In general, the defined Leatham’s media, sugar-cane medium, and beer-based media were poor substrates compared to malt extract based media. The submerged culture produced the highest amounts of biomass, followed by the surface cultures on soft agar (0.2%). But under both conditions the mycelia did not produce measurable amounts of alkaloids. The same was true for Ps. tampanensis submerged cultures in 6% malt extract with no supplement, 0.3% yeast extract, 2% pollen, or both added (data not shown).

Ps. cubensis, Ps. tampanensis, Ps. azurescens, and Ps. cyanescens were grown in different media. After 14 days the mycelium was harvested, weighed, extracted, standardized, and analyzed for psilocin and psilocybin content.
Ps. cubensis produced psilocin, but no psilocybin under the conditions analyzed. In contrast Ps. tampanensis and Ps. azurescens produced both alkaloids. Ps. azurescens and Ps. cyanescens were growing very slowly on all media tested.

Psilocin and psilocybin Rf values
Psilocin and psilocybin references (in slightly acidic solution, isolated from Ps. cyanescens fruiting bodies) were TL-chromatographed using various solvent systems. The plates were stained with Van Urk’s reagent (DMCA modification) and Rf values were observed (Table

.

Solvent system   Psilocin Rf   Psilocybin Rf
H2O: MeOH: AcOH (50 + 50 + 1)   0.92   0.88
MeOH   0.54   0.11
Acetone   0.30   0.06
MeOH: AcOH (1 + 1)   0.33   0.13
H2O   0.88   0.47
H2O: AcOH (1 + 1)   0.88   0.76
nPrOH: H2O: AcOH (10 + 3 + 3)   0.71   0.39
CH2Cl2   0.00   0.00

Best Host;
Yeast as host organism
An important factor for a succesful expression cloning procedure is the choice of an adequate host organism. Yeasts, especially S. cerevisiae, are easy to culture and grow rapidly. As well efficient transformation techniques in common with bacteria have been developed. Otherwise they have many compartments and posttranslational processing and transport systems in common with complex multicellular eukaryotes, especially with other fungi like mushrooms. For the crosstransformation from positive clones into E. coli simple methods exist (90, 84).

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   46.   The occurence of psilocybin in Gymnopilus species. G. M. Hatfield, L. J. Valdes, A. H. Smith. Lloydia 41: 140 (1978)
   47.   Verfahren zur Herstellung und Gewinnung von Psilocybin und Psilocin. R. Heim, A. Hofmann, A. Brack, H. Kobel, R. Cailleux. DBP patent 1087321 (1959)
   48.   LSD, mein Sorgenkind. A. Hofmann. Klett-Cotta, Stuttgart, Germany (1979),
      engl. transl.: LSD, my problem child. McGraw-Hill, New York (1980)
   49.   Psilocybin and Psilocin, zwei psychotrope Wirkstoffe aus mexikanischen Rauschpilzen. A. Hofmann, R. Heim, A. Brack, H. Kobel, A. Frey, H. Ott, Th. Petrzilka, F. Troxler. Helv. Chim. Acta 52: 1557 (1959)
   50.   Dephosphorylation psilocybin to psilocin by alkaline phosphatase. A. Horita, L. J. Weber. Proc. Soc. Exp. Biol. Med. 106: 32 (1961)
   51.   High efficiency transformation of Escherichia coli with plasmids. H. Inoue, H. Nojima, H. Okayama. Gene 96: 23 (1990)
   52.   Isolation of psilocybin from Psilocybe argentipes and ist determination in specimens of some mushrooms. Y. Koike, K. Wada, G. Kusano, S. Nozoe. J. Nat. Prod. 44: 362 (1981)
   53.   Cloning and characterization of two structurally and functionally divergent rhamnogalacturonases from Aspergillus aculeatus. L. V. Kofod, S. Kauppinen, S. Christgau, L. N. Andersen, H. P. Heldt-Hansen, K. Dörreich, H. Dalbøge. J. Biol. Chem. 269: 29182 (1994)
   54.   Hydroxylation of indolyl-3-acetic acid by the fungus Aspergillus niger IBFM-F-12. K. A. Koshcheenko, T. G. Baklashova, A. G. Kozlavskii, M. U. Arinbasarov, G. K. Skriabin. Prikl. Biokhim. Mikrobiol. 13: 248 (1977)
   55.   Sprühreagentien. K. G. Krebs, D. Heusser, H. Wimmer. in: Dünnschichtchromatographie, Ed. E. Stahl, Springer Verlag Berlin, Heidelberg, New York, 2. ed., p. 813 (1967)
   56.   Gymnopilus purpuratus, ein psilocybinhaltiger Pilz adventiv im Bezirk Rostock. H. Kreisel, U. Lindequist. Z. Mycol. 54: 73 (1988)
   57.   High-performance liquid chromatographic determination of some psychotropic indole derivatives. R. Kysilka, M. Wurst. J. Chrom. 21: 435 (1989)
   58.   A novel extraction procedure for psilocybin and psilocin determination in mushroom samples. R. Kysilka, M. Wurst. Planta Med. 56: 327 (1990)
   59.   H. Laatsch, Department of Organic Chemistry, University of Göttingen, Germany. preliminary communication
   60.   A chemically defined medium for the fruiting of Lentinus edodes. G. F. Leatham. Mycologia 75: 905 (1983)
   61.   Effects of growth regulating substances on fungi. K. M. Leelavathy. Can. J. Microbiol. 15: 713 (1968)
   62.   Production of psilocybin in Psilocybe baeocystis saprophytic culture. A. Y. Leung, A. H. Smith, A. G. Paul. J. Pharm. Sci. 54: 1576 (1965)
   63.   Baeocystin, a mono-methyl analog of psilocybin from Psilocybe baeocystis saprophytic culture. A. Y. Leung, A. G. Paul. J. Pharm. Sci. 56: 146 (1967)
   64.   Baeocystin and norbaeocystin: new analogs of psilocybin from Psilocybe baeocystis. A. L. Leung, A. G. Paul. J. Pharm. Sci. 57: 1667 (1968)
   65.   The relationship of carbon and nitrogen nutrition of Psilocybe baeocystis to the production of psilocybin and ist analogs. A. Y. Leung, A. G. Paul. Lloydia 32: 66 (1969)
   66.   Molecular cloning. J. Sambrook, E. F. Fritsch, T. Maniatis. Cold Spring Harbour Laboratory Press (1989)
   67.   Dextran blue as an aid for DNA precipitation and gel loading. U. Matysiak-Scholze, S. Dimmeler, M. Nehls. Elsevier Techn. Tips Online (1996)
   68.   Psilocybe semilanceata (Fr.) Quel. (Spitzkegeliger Kahlkopf). H. Michaelis. Z. Pilzkunde 43: 305 (1977)
   69.   Multiple Molecular forms of diarylpropane oxygenase, an H2O2 requiring, lignin degrading enzyme from Phanerochaete chrysosporium. V. Renganathan, K. Miki, M. H. Gold. Arch. Biochem. Biophys. 241: 304 (1985)
   70.   Permeabiliation of microorganisms by Triton X-100. G. F. Miozzari, P. Niederberger, R. Hütter. Anal. Biochem. 90: 220 (1978)
   71.   Interrelationship of phosphate nutrition, nitrogen metabolism, and accumulation of key secondary metabolites in saprophytic cultures of Psilocybe cubensis, Psilocybe cyanescens, and Paneolus campanulatus. J. M. Neal, R. G. Benedict, L. R. Brady. J. Pharm. Sci. 57: 1661 (1968)
   72.   Pharmacotheon. J. Ott. Natural Products Co., Kennewick, WA (1993)
   73.   Transformation of E. coli using homopolymer-linked plasmid chimeras. S. L. Peacock, C. M. McIver, J. J. Monahan. Biochim. Biophys. Acta 655: 243 (1981)
   74.   Determination of psilocybin in Psilocybe semilanceata by capillary zone electrophoresis. S. Pedersen-Bjergaard, E. Sannes, K. E. Rasmussen, F. Tønnesen. J. Chrom. 694: 375 (1997)
   75.   Psilocybian mycetismus with special reference to Paneolus. S. H. Pollock. J. Psyched. Drugs 8: 43 (1976)
   76.   GLC-mass spectral analysis of psilocin and psilocybin. D. B. Repke, D. T. Leslie, D. M. Mandell, N. G. Kish. J. Pharm. Sci. 66: 743 (1977)
   77.   Baeocystin in Psilocybe semilanceata. D. B. Repke. J. Pharm. Sci. 66: 113 (1977)
   78.   Baeocystin in Psilocybe, Conocybe and Paneolus. D. B. Repke. Lloydia 40: 566 (1977)
   79.   Psilocin analogs. III. Synthesis of 5-methoxy- and 5-hydroxy-1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indoles. D. B. Repke, W. J. Ferguson. J. Het. Chem. 19: 845 (1982)
   80.   Site of action of growth inhibitory tryptophan analogues in Catharanthus roseus cell suspension cultures. F. Sasse, M. Buchholz, J. Berlin. Z. Naturforsch. 38c: 910, 916 (1983)
   81.   Pihkal. A. Shulgin, A. Shulgin. Transform Press, Berkeley, CA (1991)
   82.   Tihkal. A. Shulgin, A. Shulgin. Transform Press, Berkeley, CA (1997)
   83.   Psilocybin in Fruchtkörpern von Inocybe aeruginascens. M. Smerdzieva, M. Wurst, T. Koza, J. Gartz. Planta Med. 83 (1986)
   84.   A rapid and inexpensive method for isolation of shuttle vector DNA from yeast for the transformation of E. coli. R. Soni, J. A. H. Murray. Nucl. Acid. Res. 20: 5852 (1992)
   85.   A highly efficient directional cDNA cloning method utilizing an asymmetrically tailed linker-primer plasmid. N. Spickofsky, R. F. Margolskee. Nucl. Acid Res. 19: 7105 (1991)
   86.   The mushroom cultivator. P. Stamets, J. S. Chilton. Agarikon Press, Olympia, WA (1983)
   87.   Occurence of 5-hydroxylated indole derivatives in Paneolina foenescii (Fries) Kuehner from various origin. T. Stijve, C. Hischenhuber, D. Ashley. Z. Mycol. 50: 361 (1984)
   88.   Psilocin, psilocybin, serotonin and urea in Paneolus cyanescens from various origin. T. Stijve. Persoonia 15: 117 (1992)
   89.   Convenient and effective methods for in vitro cultivation of mycelium and fruiting bodies of Lentinus edodes. Y. H. Tan, D. Moore. Mycol. Res. 96: 14077 (1992)
   90.   A simple method for rescuing autonomous plasmids from fission yeast. A. Topal, S. Karaer, G. Temizkan. Elsevier Techn. Tips Online (97)
   91.   Basic yeast methods. J. H. Toyn. Meth. Mol. Cell. Biol. 5: 249 (1995)
   92.   Influence of plant hormones on a wood-rotting fungus, Coriolus versicolor. S. I. Tsujiyama, J. I. Azuma, K. Okamura. Transact. Mycol. Soc. Japan 34: 369
   93.   Occurence of serotonin in a hallucinogenic mushroom. V. E. Tyler, JR. Science. 128: 718 (1958)
   94.   Exogenous regulators in the mycelium of Pleurotus ostreatus after exogenous application. K. Vinklarkova, Z. Sladky. Folia Microbiol. Praha 23: 55 (1978)
   95.   Mushrooms, Russia and history. Volumes 1 and 2. V. P. Wasson, R. G. Wasson. Pantheon Books, New York (1957)
   96.   A new psilocybian species of Copelandia. R. A. Weeks. J. Nat. Prod. 42: 469 (1979)
   97.   Analysis of psychotropic compounds in fungi of the genus Psilocybe by reversed phase high-performance liquid chromatography. M. Wurst, M. Semerdzieva, J. Vokoun. J. Chrom. 286: 229 (1984)
   98.   Analysis and isolation of indole alkaloids of fungi by high-performance liquid chromatography. M. Wurst, R. Kysilka, T. Koza. J. Chrom. 593: 201 (1992)

Quicksilver · « **Reply #12 on:** February 20, 2001, 03:47:00 PM »

Bioboy,
Thanks for the ref! What do you think the odds of this enzyme working on a more simple compound (tryptamine) compared to the one the authors used (desacetoxyvindoline) ??

Don't suppose anybody has molecular ligand-binding software they want to share? (Windows or MacOS)
(e.g. Sybyl, Autodock, Insight ...but those are all unix i think??)

-quicksilver-

stormwind · « **Reply #13 on:** March 03, 2001, 12:06:00 AM »

Man Oh Man - when you guys get these yeasts bred and multiplied, please let J***F or someone market some - I'm kinda short on realchem practical knowledge, but I could handle some sourdough starter without blowing the house up I think...

"I held my nose I closed my eyes
I took a drink
I didn't know if it was day or night..."

ZyGoat · « **Reply #14 on:** March 03, 2001, 05:36:00 AM »

This is probably a stupid question, but why go to all the trouble of creating a transgenic psilocin producing yeast/bacteria when there already exists a load of fungal organisms capable of this?

stormwind · « **Reply #15 on:** March 03, 2001, 03:17:00 PM »

yeast is cheap
yeast is easy to grow
yeast works fast
A neat culture might be a big barrel held in the dark at the correct temp
theoretical end products are easily extracted
yeast isn't banned anywhere, all strains are as far as FOAF knows universally legal...
even if some were not it would take a microbiologist to tell which ones, IF he could without DNA sequencing...
And bread is always gonna be OTC, is how this non-chemist hick sees it...so might be useful to legitimate researchers for production of theoretical legal compounds...
Man I am gonna go find a home brewers site to study...
Hows this sound, "Stinging Bee Rainbow Beer, don't drink too much, or you'll bee up all night!"

Teonanacatl · « **Reply #16 on:** March 24, 2001, 01:42:00 PM »

About the inhibition of INMT...best thing would be to do an enzyme assay...chances are, the DMT is acting as a competetive inhibitor...however, if by chance DMT is acting non or uncompetetively, mutations to the E may remove this aspect of control....INMT from rabbit lung has the following relative activities with varying substrate:
alpha-methyltryptamine: 131%
tryptamine: 100%
norharmane: 58%
N-methyltryptamine: 44%
serotonin: 11%
beta-phenethylamine: 5%
desipramine: 4%
nortryptyline/1,2,3,4-tetrahydroisoquinoline/phenethanolamine: 3%
tyramine/(+/-)octopamine: 1%
ethylamine: 0.5%
all others are <0.5% (histamine, DMT, dopamine, epinephrine, epinine, ethanolamine, harmane-1,2,3,4-tetrahydrocarboxylic acid, harmol, melatonin, methylamine, nicotinamide, norepinephrine, salsolinol)
Note - DMT does have very low action...also points to competetive inhibition...the problem of DMT inhibiting INMT may be dealt with in other manners too...some working in the tissue culture field have come across the problem of producing alkaloids in plant material as it tends to build up toxic levels of alkaloids the plants can't handle...one method of dealing with this is to make the cells more open to the environment or to cause them to release the final products into solution...this has been done with cell damage (which also tends to elicit 2° metabolism, but tendst to slow down later growth), application of low levels of H2O2 as well as a number of synthetic compounds I can't recall right now, I think they were PEGs (polyethyleneglycols)...
I believe the thesis lilienthal gave you is very helpful, however, cloning of the E's in the pathway was not completed...another factor which must be looked at is the regulation of each of the genes...yeast is nice for this cause it tends to express genes from higher fungi with no problem...remove the gene with the 5' region attached and look for regulatory factors...or if you're lazy, remove the gene, cut off the 5' region and attach a strong, constitutively expressed promoter (ie. from 1° metabolism)...voila, lots o' E's

Begin with the dissolution of superfluous matters
So that desire and consciousness are free

Teonanacatl · « **Reply #17 on:** March 24, 2001, 02:01:00 PM »

Hey KrZ...I just did a translated blast search (protein-->nucleotide)...lots o' hits, your second sequence tended to have higher homology with INMTs...some are just general aromatic amino acid decarboxylases, some hits were also for the similar Tyr/Dopa decarboxylases, which are specific to these two substrates (eg. from Papaver somniferum)...you should also consider up-regulating production of S-adenosylmethionine if you are going to express the INMT at high levels, otherwise it might become a limiting factor in the reaction...anyways, here's the top hits(1st 5 from your first sequence, 2nd 5 from your second AA sequence)...now just look for conserved sequences between the enzymes that you are most interested in and see if there are any losses of this conservation in the Tyr/Dopa decarboxylases...hope this helps:

1: P17770 AROMATIC-L-AMINO-ACID DECARBOXYLASE (DOPA DECARBOXYLASE) (TRYPTOPHAN DECARBOXYLASE) BLink, PubMed, Related Sequences, Taxonomy, LinkOut

LOCUS    DCD_CATRO    500 aa PLN    01-NOV-1995
DEFINITION AROMATIC-L-AMINO-ACID DECARBOXYLASE (DOPA DECARBOXYLASE)
(TRYPTOPHAN DECARBOXYLASE).
ACCESSION   P17770
PID    g118306
VERSION    P17770 GI:118306
DBSOURCE swissprot: locus DCD_CATRO, accession P17770;
class: standard.
created: Aug 1, 1990.
sequence updated: Aug 1, 1990.
annotation updated: Nov 1, 1995.
xrefs: gi: gi: 167489, gi: gi: 167490, gi: gi: 18225, gi: gi:
18226, gi: gi: 1247291, gi: gi: 1247292, gi: gi: 68027, gi: gi:
100170, gi: gi: 542023
xrefs (non-sequence databases): PFAM PF00282, PROSITE PS00392
KEYWORDS Lyase; Decarboxylase; Pyridoxal phosphate.
SOURCE Madagascar periwinkle.
ORGANISM Catharanthus roseus
Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
euphyllophytes; Spermatophyta; Magnoliophyta; eudicotyledons; core
eudicots; Asteridae; euasterids I; Gentianales; Apocynaceae;
Catharanthus.
REFERENCE   1 (residues 1 to 500)
AUTHORS   De Luca,V., Marineau,C. and Brisson,N.
TITLE    Molecular cloning and analysis of cDNA encoding a plant tryptophan
decarboxylase: comparison with animal dopa decarboxylases
JOURNAL   Proc. Natl. Acad. Sci. U.S.A. 86 (

, 2582-2586 (1989)
MEDLINE   89202373
REMARK SEQUENCE FROM N.A.
REFERENCE   2 (residues 1 to 500)
AUTHORS   Goddijn,O.J., Lohman,F.P., de Kam,R.J., Schilperoort,R.A. and
Hoge,J.H.
TITLE    Nucleotide sequence of the tryptophan decarboxylase gene of
Catharanthus roseus and expression of tdc-gusA gene fusions in
Nicotiana tabacum
JOURNAL   Mol. Gen. Genet. 242 (2), 217-225 (1994)
MEDLINE   94211212
REMARK SEQUENCE FROM N.A.
STRAIN=CV. MORNING MIST; TISSUE=LEAF
COMMENT    -------------------------------------------------------------------
This SWISS-PROT entry is copyright. It is produced through a
collaboration between the Swiss Institute of Bioinformatics and
the EMBL outstation - the European Bioinformatics Institute.
The original entry is available from

http://www.expasy.ch/sprot

and

http://www.ebi.ac.uk/sprot

------------------------------------------------------------------.
[CATALYTIC ACTIVITY] L-TRYPTOPHAN = TRYPTAMINE + CO(2) (ALSO ACTS
ON 5-HYDROXY-L-TRYPTOPHAN AND DIHYDROXY-L-PHENYLALANINE (DOPA)).
[COFACTOR] PYRIDOXAL PHOSPHATE.
[SUBUNIT] HOMODIMER.
[SIMILARITY] BELONGS TO GROUP II DECARBOXYLASES (DDC, GAD, HDC AND
TYRDC).
FEATURES    Location/Qualifiers
   source 1..500
   /organism="Catharanthus roseus"
   /db_xref="taxon:4058"
   1..500
   Protein    1..500
   /product="AROMATIC-L-AMINO-ACID DECARBOXYLASE"
   /EC_number="4.1.1.28"
   Site 319
   /site_type="binding"
   /note="PYRIDOXAL PHOSPHATE (BY SIMILARITY)."
ORIGIN
1 mgsidstnva msnspvgefk pleaeefrkq ahrmvdfiad yyknvetypv lsevepgylr
   61 kripetapyl peplddimkd iqkdiipgmt nwmspnfyaf fpatvssaaf lgemlstaln
121 svgftwvssp aatelemivm dwlaqilklp ksfmfsgtgg gviqnttses ilctiiaare
181 raleklgpds igklvcygsd qthtmfpktc klagiypnni rlipttvetd fgispqvlrk
241 mveddvaagy vplflcatlg ttsttatdpv dslseianef giwihvdaay agsacicpef
301 rhyldgierv dslslsphkw llayldctcl wvkqphlllr alttnpeylk nkqsdldkvv
361 dfknwqiatg rkfrslklwl ilrsygvvnl qshirsdvam gkmfeewvrs dsrfeivvpr
421 nfslvcfrlk pdvsslhvee vnkklldmln stgrvymtht ivggiymlrl avgsslteeh
481 hvrrvwdliq kltddllkea
//

1: AAB39709 tryptophan decarboxylase [Camptotheca acuminata] BLink, Related Sequences, Nucleotide, Taxonomy

LOCUS    AAB39709 498 aa PLN    02-JAN-1997
DEFINITION tryptophan decarboxylase [Camptotheca acuminata].
ACCESSION   AAB39709
PID    g1763279
VERSION    AAB39709.1 GI:1763279
DBSOURCE locus CAU73657 accession U73657.1
KEYWORDS .
SOURCE Camptotheca acuminata.
ORGANISM Camptotheca acuminata
Eukaryota; Viridiplantae; Embryophyta; Tracheophyta; Spermatophyta;
Magnoliophyta; eudicotyledons; core eudicots; Asteridae; Cornales;
Cornaceae; Nyssoideae; Camptotheca.
REFERENCE   1 (residues 1 to 498)
AUTHORS   Lopez-Meyer,M. and Nessler,C.L.
TITLE    Tryptophan decarboxylase is encoded by two autonomously regulated
genes in Camptotheca acuminata which are differentially expressed
during development and stress
JOURNAL   Unpublished
REFERENCE   2 (residues 1 to 498)
AUTHORS   Lopez-Meyer,M. and Nessler,C.L.
TITLE    Direct Submission
JOURNAL   Submitted (08-OCT-1996) Biology, Texas A&M University, College
Station, TX 77843-3258, USA
FEATURES    Location/Qualifiers
   source 1..498
   /organism="Camptotheca acuminata"
   /db_xref="taxon:16922"
   /tissue_type="leaf"
   Protein    1..498
   /function="converts tryptohan to tryptamine"
   /product="tryptophan decarboxylase"
   /EC_number="4.1.1.28"
   CDS    1..498
   /gene="tdc2"
   /coded_by="U73657.1:1576..3072"
   /note="TDC"
ORIGIN
1 mgsidsnydt esagqcrple peefrkqahq mvdfiadyyk niesypvlsq vepgylqsrl
   61 petapyrpep fesilkdvhk diipgvthwl spnffayfpa tvssaafvge mlctcfnavg
121 fnwlaspael elemvvmdwl asmlklpnsf tflgtgggvi qgttseailc tliaardral
181 esigvdsihk lvvygsdqth styakacnla gilpcnirsi rteavanfsl spdslhreie
241 advaagmvpl ylcatvgtts ttaidslspl advandyglw fhvdaayags acicpefrhy
301 ldgieradsl slsphkwlls yldccclwvk rpsvlvkals tdpeylknkp sesnsvvdfk
361 dwqvgtgrrf kalrlwfvmr sygvanlqsh irsdiqmakm feefvnsdpr feivvprvfs
421 lvcfrlnpfs ksdpcntell nrkllewvns tgqvyithtk vggvymlrfa vgatlteehh
481 vsaawklire gadallcs
//

1: AAB39708 tryptophan decarboxylase [Camptotheca acuminata] BLink, Related Sequences, Nucleotide, Taxonomy

LOCUS    AAB39708 502 aa PLN    02-JAN-1997
DEFINITION tryptophan decarboxylase [Camptotheca acuminata].
ACCESSION   AAB39708
PID    g1763277
VERSION    AAB39708.1 GI:1763277
DBSOURCE locus CAU73656 accession U73656.1
KEYWORDS .
SOURCE Camptotheca acuminata.
ORGANISM Camptotheca acuminata
Eukaryota; Viridiplantae; Embryophyta; Tracheophyta; Spermatophyta;
Magnoliophyta; eudicotyledons; core eudicots; Asteridae; Cornales;
Cornaceae; Nyssoideae; Camptotheca.
REFERENCE   1 (residues 1 to 502)
AUTHORS   Lopez-Meyer,M. and Nessler,C.L.
TITLE    Tryptophan decarboxylase is encoded by two autonomously regulated
genes in Camptotheca acuminata which are differentially expressed
during development and stress
JOURNAL   Unpublished
REFERENCE   2 (residues 1 to 502)
AUTHORS   Lopez-Meyer,M. and Nessler,C.L.
TITLE    Direct Submission
JOURNAL   Submitted (08-OCT-1996) Biology, Texas A&M University, College
Station, TX 77843-3258, USA
FEATURES    Location/Qualifiers
   source 1..502
   /organism="Camptotheca acuminata"
   /db_xref="taxon:16922"
   /dev_stage="7-day-old seedlings"
   Protein    1..502
   /function="converts tryptophan into tryptamine"
   /product="tryptophan decarboxylase"
   /EC_number="4.1.1.28"
   CDS    1..502
   /gene="tdc1"
   /coded_by="U73656.1:189..1697"
   /note="TDC"
ORIGIN
1 mgsldsnydt espasvgqfn pldpeefrkq ahcivdfiad yykniesypv lsqvdpgyrh
   61 srlgknapyr sepfesilkd vqkdiipgmt hwmspnffah fpatvssaaf vgemlctcfn
121 svgfnwlasp aatelemvvi dwlanmlklp ksfmfsgtgg gvlqgttsea ilctliaasp
181 mhfeivgvkt stsfvvygsd qthstyakac klagilpcni rsipttadsn fsvsplllrr
241 aieadkaagm vplyicatvg ttsttaidpl ssladvandy gvwfhvdaay agsacicpef
301 rhyldgiera dslslsphkw llsyldcccl wvkspsllvk alstdpeylk nqpsesksvv
361 dykdwqvgtg rrfkalrlwf vmrsygvanl qshirtdvqm akmfegfvks dprfeilvpr
421 vfslvcfrln pisgsdptgt ealnrklldw vnstgrvymt htkvggiyml rfavgatlte
481 krhvssawkl ikegadvllk ed
//

1: AAG60665 tyrosine/dopa decarboxylase [Thalictrum flavum subsp. glaucum] BLink, Related Sequences, Nucleotide, Taxonomy

LOCUS    AF314150_1 518 aa PLN    31-JAN-2001
DEFINITION tyrosine/dopa decarboxylase [Thalictrum flavum subsp. glaucum].
ACCESSION   AAG60665
PID    g12620328
VERSION    AAG60665.1 GI:12620328
DBSOURCE locus AF314150 accession AF314150.1
KEYWORDS .
SOURCE Thalictrum flavum subsp. glaucum.
ORGANISM Thalictrum flavum subsp. glaucum
Eukaryota; Viridiplantae; Embryophyta; Tracheophyta; Spermatophyta;
Magnoliophyta; eudicotyledons; Ranunculales; Ranunculaceae;
Thalictrum.
REFERENCE   1 (residues 1 to 518)
AUTHORS   Samanani,N. and Facchini,P.J.
TITLE    Cloning and characterization of a tyrosine/dopa decarboxylase cDNA
from the meadow rue, Thalictrum flavum ssp. glaucum
JOURNAL   Unpublished
REFERENCE   2 (residues 1 to 518)
AUTHORS   Samanani,N. and Facchini,P.J.
TITLE    Direct Submission
JOURNAL   Submitted (16-OCT-2000) Department of Biological Sciences,
University of Calgary, 2500 University Drive N.W., Calgary, Alberta
T2N 1N4, Canada
COMMENT    Method: conceptual translation supplied by author.
FEATURES    Location/Qualifiers
   source 1..518
   /organism="Thalictrum flavum subsp. glaucum"
   /sub_species="glaucum"
   /db_xref="taxon:150095"
   /note="cell suspension culture"
   Protein    1..518
   /product="tyrosine/dopa decarboxylase"
   /EC_number="4.1.1.25"
   CDS    1..518
   /gene="TYDC1"
   /coded_by="AF314150.1:116..1672"
ORIGIN
1 mgslhvedld niskctvenp ldpeefrrqg hmmidflady yrdiekypvr sqvepgylrk
   61 eipdsapynp esietiledv hkqiipgith wqspnyfayf pssgsvagfl gemlstgfnv
121 vgfnwmsspa atelesivmd wlgkmlklpk sflfsgnggg vlqgttceai lctltaardr
181 mlnkigreni cklvvygsdq thcalqkaaq iagihpnnfr avpttkandy glsasalrst
241 iledieaglv plflcatvgt tsstavdpig plckvasdys iwvhvdaaya gsacicpefr
301 hfidgvenad sfslnahkwf fttldccclw vkepsalika lstnpeylrn kateshqvvd
361 ykdwqialsr rframklwlv lrsygvanlr nflrshvkma knfegfiald krfeivvprt
421 famvcfrllp prspliiktn gyqngngvyh kdesraneln rrllesinas gsaymthsmv
481 ggvymirfav gaslteerhv ilawkvvqeh adavlatf
//

1: P54769 TYROSINE/DOPA DECARBOXYLASE 2 [INCLUDES: DOPA DECARBOXYLASE (DDC); TYROSINE DECARBOXYLASE ] BLink, PubMed, Related Sequences, Taxonomy, LinkOut

LOCUS    TYD2_PAPSO 531 aa PLN    15-JUL-1999
DEFINITION TYROSINE/DOPA DECARBOXYLASE 2 [INCLUDES: DOPA DECARBOXYLASE (DDC);
TYROSINE DECARBOXYLASE ].
ACCESSION   P54769
PID    g1717826
VERSION    P54769 GI:1717826
DBSOURCE swissprot: locus TYD2_PAPSO, accession P54769;
class: standard.
created: Oct 1, 1996.
sequence updated: Oct 1, 1996.
annotation updated: Jul 15, 1999.
xrefs: gi: gi: 607746, gi: gi: 607747
xrefs (non-sequence databases): PFAM PF00282, PROSITE PS00392
KEYWORDS Lyase; Decarboxylase; Pyridoxal phosphate; Multigene family.
SOURCE opium poppy.
ORGANISM Papaver somniferum
Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
euphyllophytes; Spermatophyta; Magnoliophyta; eudicotyledons;
Ranunculales; Papaveraceae; Papaver.
REFERENCE   1 (residues 1 to 531)
AUTHORS   Facchini,P.J. and De Luca,V.
TITLE    Differential and tissue-specific expression of a gene family for
tyrosine/dopa decarboxylase in opium poppy
JOURNAL   J. Biol. Chem. 269 (43), 26684-26690 (1994)
MEDLINE   95014524
REMARK SEQUENCE FROM N.A.
STRAIN=CV. MARIANNE
COMMENT    -------------------------------------------------------------------
This SWISS-PROT entry is copyright. It is produced through a
collaboration between the Swiss Institute of Bioinformatics and
the EMBL outstation - the European Bioinformatics Institute.
The original entry is available from

http://www.expasy.ch/sprot

and

http://www.ebi.ac.uk/sprot

------------------------------------------------------------------.
[FUNCTION] MARGINALLY HIGHER SUBSTRATE SPECIFICITY FOR L-DOPA OVER
L-TYROSINE.
[CATALYTIC ACTIVITY] L-TYROSINE = TYRAMINE + CO(2).
[COFACTOR] PYRIDOXAL PHOSPHATE.
[SUBUNIT] HOMODIMER (BY SIMILARITY).
[TISSUE SPECIFICITY] PREDOMINANTLY EXPRESSED IN THE ROOTS AND
STEMS, WHILE A LOWER LEVEL EXPRESSION IS SEEN IN THE SEPALS AND
CARPELS OF FULLY EXPANDED FLOWERS.
[SIMILARITY] BELONGS TO GROUP II DECARBOXYLASES (DDC, GAD, HDC AND
TYRDC).
FEATURES    Location/Qualifiers
   source 1..531
   /organism="Papaver somniferum"
   /db_xref="taxon:3469"
   1..531
   Protein    1..531
   /product="TYROSINE/DOPA DECARBOXYLASE 2 [INCLUDES: DOPA
   DECARBOXYLASE"
   /EC_number="4.1.1.28"
   /EC_number="4.1.1.25"
   Site 319
   /site_type="binding"
   /note="PYRIDOXAL PHOSPHATE (BY SIMILARITY)."
ORIGIN
1 mgslntedvl enssafgvtn pldpeefrrq ghmiidflad yyrdvekypv rsqvepgylr
   61 krlpetapyn pesietilqd vtteiipglt hwqspnyyay fpssgsvagf lgemlstgfn
121 vvgfnwmssp aatelesvvm dwfgkmlnlp esflfsgsgg gvlqgtscea ilctltaard
181 rklnkigreh igrlvvygsd qthcalqkaa qvaginpknf raiktfkens fglsaatlre
241 viledieagl iplfvcptvg ttsstavdpi spicevakey emwvhvdaay agsacicpef
301 rhfidgveea dsfslnahkw ffttldcccl wvkdpsalvk alstnpeylr nkatesrqvv
361 dykdwqials rrfrslklwm vlrsygvtnl rnflrshvkm aktfeglicm dgrfeitvpr
421 tfamvcfrll ppktikvydn gvhqngngvv plrdenenlv lanklnqvyl etvnatgsvy
481 mthavvggvy mirfavgstl teerhviyaw kilqehadli lgkfseadfs s
//

1: AAC97491 indolethylamine N-methyltransferase [Oryctolagus cuniculus] BLink, PubMed, Related Sequences, Nucleotide, Taxonomy

LOCUS    AAC97491 263 aa MAM    22-DEC-1998
DEFINITION indolethylamine N-methyltransferase [Oryctolagus cuniculus].
ACCESSION   AAC97491
PID    g4039109
VERSION    AAC97491.1 GI:4039109
DBSOURCE locus AF077826 accession AF077826.1
KEYWORDS .
SOURCE rabbit.
ORGANISM Oryctolagus cuniculus
Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;
Mammalia; Eutheria; Lagomorpha; Leporidae; Oryctolagus.
REFERENCE   1 (residues 1 to 263)
AUTHORS   Thompson,M.A. and Weinshilboum,R.M.
TITLE    Rabbit lung indolethylamine N-methyltransferase. cDNA and gene
cloning and characterization
JOURNAL   J. Biol. Chem. 273 (51), 34502-34510 (1998)
MEDLINE   99069450
REFERENCE   2 (residues 1 to 263)
AUTHORS   Thompson,M.A. and Weinshilboum,R.M.
TITLE    Direct Submission
JOURNAL   Submitted (13-JUL-1998) Pharmacology, Mayo Clinic/Mayo Foundation,
200 1st St. SW, Rochester, MN 55905, USA
COMMENT    Method: conceptual translation supplied by author.
FEATURES    Location/Qualifiers
   source 1..263
   /organism="Oryctolagus cuniculus"
   /db_xref="taxon:9986"
   /tissue_type="lung"
   Protein    1..263
   /function="catalyzes the N-methylation of tryptamine"
   /product="indolethylamine N-methyltransferase"
   CDS    1..263
   /gene="INMT"
   /coded_by="AF077826.1:16..807"
ORIGIN
1 meggftggde yqkhflprdy lntyysfqsg pspeaemlkf nleclhktfg pgglqgdtli
   61 digsgptiyq vlaacesfkd itlsdftdrn reelakwlkk epgaydwtpa lkfacelegn
121 sgrwqekaek lratvkrvlk cdanlsnplt pvvlppadcv ltllamecac csldayraal
181 rnlasllkpg ghlvttvtlq lssymvgere fscvalekee veqavldagf dieqllyspq
241 sysastapnr gvcflvarkk pgs
//

1: XP_004862 indolethylamine N-methyltransferase [Homo sapiens] BLink, Related Sequences, Nucleotide, Taxonomy, LinkOut

LOCUS    XP_004862    263 aa PRI    09-FEB-2001
DEFINITION indolethylamine N-methyltransferase [Homo sapiens].
ACCESSION   XP_004862
PID    g11420979
VERSION    XP_004862.1 GI:11420979
DBSOURCE REFSEQ: accession XM_004862.1
KEYWORDS .
SOURCE human.
ORGANISM Homo sapiens
Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;
Mammalia; Eutheria; Primates; Catarrhini; Hominidae; Homo.
REFERENCE   1 (residues 1 to 263)
AUTHORS   NCBI Annotation Project.
TITLE    Direct Submission
JOURNAL   Submitted (04-FEB-2001) National Center for Biotechnology
Information, NIH, Bethesda, MD 20894, USA
COMMENT    GENOME ANNOTATION REFSEQ: This reference sequence was derived by
automated computational analysis of NCBI genomic sequence contig
NT_007825 using gene prediction method: Acembly.
Supporting evidence includes similarity to: 71 proteins, 1 mRNAs
See details in AceView
Method: conceptual translation supplied by author.
FEATURES    Location/Qualifiers
   source 1..263
   /organism="Homo sapiens"
   /db_xref="taxon:9606"
   /chromosome="7"
   Protein    1..263
   /product="indolethylamine N-methyltransferase"
   Region 4..259
   /region_name="NNMT/PNMT/TEMT family"
   /db_xref="CDD:pfam01234"
   /note="NNMT_PNMT_TEMT"
   CDS    1..263
   /gene="INMT"
   /db_xref="LocusID:11185"
   /db_xref="MIM:604854"
   /coded_by="TR00067273:1..792"
ORIGIN
1 mkggftggde yqkhflprdy latyysfdgs pspeaemlkf nleclhktfg pgglqgdtli
   61 digsgptiyq vlaacdsfqd itlsdftdrn reelekwlkk epgaydwtpa vkfacelegn
121 sgrweekeek lraavkrvlk cdvhlgnpla pavlpladcv ltllamecac csldayraal
181 cnlasllkpg ghlvttvtlr lpsymvgkre fscvalekee veqavldagf dieqllhspq
241 sysvtnaann gvcfivarkk pgp
//

1: NP_006765 indolethylamine N-methyltransferase; thioester S-methyltransferase-like [Homo sapiens] BLink, PubMed, Related Sequences, Nucleotide, Taxonomy, OMIM, LinkOut

LOCUS    NP_006765    263 aa PRI    03-FEB-2001
DEFINITION indolethylamine N-methyltransferase; thioester
S-methyltransferase-like [Homo sapiens].
ACCESSION   NP_006765
PID    g10092584
VERSION    NP_006765.2 GI:10092584
DBSOURCE REFSEQ: accession NM_006774.2
KEYWORDS .
SOURCE human.
ORGANISM Homo sapiens
Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;
Mammalia; Eutheria; Primates; Catarrhini; Hominidae; Homo.
REFERENCE   1 (residues 1 to 263)
AUTHORS   Thompson,M.A., Moon,E., Kim,U.J., Xu,J., Siciliano,M.J. and
Weinshilboum,R.M.
TITLE    Human indolethylamine N-methyltransferase: cDNA cloning and
expression, gene cloning, and chromosomal localization
JOURNAL   Genomics 61 (3), 285-297 (1999)
MEDLINE   20021766
   PUBMED   10552930
COMMENT    PROVISIONAL REFSEQ: This record has not yet been subject to final
NCBI review. The reference sequence was derived from AF128848.1,
AF128847.1.
On Sep 12, 2000 this sequence version replaced gi:5803044.
FEATURES    Location/Qualifiers
   source 1..263
   /organism="Homo sapiens"
   /db_xref="taxon:9606"
   /chromosome="7"
   /map="7p15.3-p15.2"
   Protein    1..263
   /product="indolethylamine N-methyltransferase"
   /note="thioester S-methyltransferase-like"
   Region 4..259
   /region_name="NNMT/PNMT/TEMT family"
   /db_xref="CDD:pfam01234"
   /note="NNMT_PNMT_TEMT"
   CDS    1..263
   /gene="INMT"
   /db_xref="LocusID:11185"
   /db_xref="MIM:604854"
   /coded_by="NM_006774.2:1..792"
ORIGIN
1 mkggftggde yqkhflprdy latyysfdgs pspeaemlkf nleclhktfg pgglqgdtli
   61 digsgptiyq vlaacdsfqd itlsdftdrn reelekwlkk epgaydwtpa vkfacelegn
121 sgrweekeek lraavkrvlk cdvhlgnpla pavlpladcv ltllamecac csldayraal
181 cnlasllkpg ghlvttvtlr lpsyvvgkre fscvalekee veqavldagf dieqllhspq
241 sysvtnaann gvccivarkk pgp
//

1: AAF18305 indolethylamine N-methyltransferase [Homo sapiens] BLink, PubMed, Related Sequences, Nucleotide, Taxonomy, OMIM, LinkOut

LOCUS    AF128847_1 263 aa PRI    16-DEC-1999
DEFINITION indolethylamine N-methyltransferase [Homo sapiens].
ACCESSION   AAF18305
PID    g6580817
VERSION    AAF18305.1 GI:6580817
DBSOURCE locus AF128847 accession AF128847.1
KEYWORDS .
SOURCE human.
ORGANISM Homo sapiens
Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;
Mammalia; Eutheria; Primates; Catarrhini; Hominidae; Homo.
REFERENCE   1 (residues 1 to 263)
AUTHORS   Thompson,M.A., Moon,E., Kim,U.J., Xu,J., Siciliano,M.J. and
Weinshilboum,R.M.
TITLE    Human indolethylamine N-methyltransferase: cDNA cloning and
expression, gene cloning, and chromosomal localization
JOURNAL   Genomics 61 (3), 285-297 (1999)
MEDLINE   20021766
   PUBMED   10552930
REFERENCE   2 (residues 1 to 263)
AUTHORS   Thompson,M.A., Moon,E., Kim,U.-J., Xu,J., Siciliano,M.J. and
Weinshilboum,R.M.
TITLE    Direct Submission
JOURNAL   Submitted (17-FEB-1999) Pharmacology, Mayo Clinic, 200 1st St. SW,
Rochester, MN 55905, USA
COMMENT    Method: conceptual translation supplied by author.
FEATURES    Location/Qualifiers
   source 1..263
   /organism="Homo sapiens"
   /db_xref="taxon:9606"
   /chromosome="7"
   /map="7p15.2-p15.3"
   Protein    1..263
   /product="indolethylamine N-methyltransferase"
   /name="INMT"
   CDS    1..263
   /gene="INMT"
   /coded_by="AF128847.1:17..808"
ORIGIN
1 mkggftggde yqkhflprdy latyysfdgs pspeaemlkf nleclhktfg pgglqgdtli
   61 digsgptiyq vlaacdsfqd itlsdftdrn reelekwlkk epgaydwtpa vkfacelegn
121 sgrweekeek lraavkrvlk cdvhlgnpla pavlpladcv ltllamecac csldayraal
181 cnlasllkpg ghlvttvtlr lpsymvgkre fscvalekge veqavldagf dieqllhspq
241 sysvtnaann gvccivarkk pgp
//

1: BAB28594 putative [Mus musculus] BLink, PubMed, Related Sequences, Nucleotide, Taxonomy, LinkOut

LOCUS    BAB28594 264 aa ROD    08-FEB-2001
DEFINITION putative [Mus musculus].
ACCESSION   BAB28594
PID    g12850108
VERSION    BAB28594.1 GI:12850108
DBSOURCE locus AK013010 accession AK013010.1
KEYWORDS .
SOURCE house mouse.
ORGANISM Mus musculus
Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;
Mammalia; Eutheria; Rodentia; Sciurognathi; Muridae; Murinae; Mus.
REFERENCE   1 (sites)
AUTHORS   Carninci,P. and Hayashizaki,Y.
TITLE    High-efficiency full-length cDNA cloning
JOURNAL   Methods Enzymol. 303, 19-44 (1999)
REFERENCE   2 (sites)
AUTHORS   Carninci,P., Shibata,Y., Hayatsu,N., Sugahara,Y., Shibata,K.,
Itoh,M., Konno,H., Okazaki,Y., Muramatsu,M. and Hayashizaki,Y.
TITLE    Normalization and subtraction of cap-trapper-selected cDNAs to
prepare full-length cDNA libraries for rapid discovery of new genes
JOURNAL   Genome Res. 10 (10), 1617-1630 (2000)
MEDLINE   20499374
REFERENCE   3 (sites)
AUTHORS   Shibata,K., Itoh,M., Aizawa,K., Nagaoka,S., Sasaki,N., Carninci,P.,
Konno,H., Akiyama,J., Nishi,K., Kitsunai,T., Tashiro,H., Itoh,M.,
Kikuchi,N., Ishii,Y., Nakamura,S., Hazama,M., Nishine,T.,
Harada,A., Yamamoto,R., Matsumoto,H., Sakaguchi,S., Ikegami,T.,
Kashiwagi,K., Fujiwake,S., Inoue,K., Togawa,Y., Izawa,M., Ohara,E.,
Watahiki,M., Yoneda,Y., Ishikawa,T., Ozawa,K., Tanaka,T.,
Matsuura,S., Okazaki,Y., Muramatsu,M., Inoue,Y. and Hayashizaki,Y.
TITLE    RIKEN integrated sequence analysis (RISA) system--384-format
sequencing pipeline with 384 multicapillary sequencer
JOURNAL   Genome Res. 10 (11), 1757-1771 (2000)
MEDLINE   20530913
REFERENCE   4 (sites)
AUTHORS   The RIKEN Genome Exploration Research Group Phase II Team and
FANTOM Consortium.
TITLE    Functional annotation of a full-length mouse cDNA collection
JOURNAL   Nature 409, 685-690 (2001)
REFERENCE   5 (residues 1 to 264)
AUTHORS   Adachi,J., Aizawa,K., Akahira,S., Akimura,T., Aono,H., Arai,A.,
Arakawa,T., Carninci,P., Fukuda,S., Fukunishi,Y., Furuno,M.,
Hanagaki,T., Hara,A., Hayatsu,N., Hiramoto,K., Hiraoka,T., Hori,F.,
Imotani,K., Ishii,Y., Itoh,M., Izawa,M., Kato,H., Kawai,J.,
Kojima,Y., Konno,H., Kouda,M., Koya,S., Kurihara,C., Matsuyama,T.,
Miyazaki,A., Nishi,K., Nomura,K., Numazaki,R., Ohno,M., Okazaki,Y.,
Okido,T., Owa,C., Saito,H., Saito,R., Sakai,C., Sakai,K., Sano,H.,
Sasaki,D., Shibata,K., Shibata,Y., Shinagawa,A., Shiraki,T.,
Sogabe,Y., Suzuki,H., Tagami,M., Tagawa,A., Takahashi,F.,
Tanaka,T., Tejima,Y., Toya,T., Yamamura,T., Yasunishi,A.,
Yoshida,K., Yoshino,M., Muramatsu,M. and Hayashizaki,Y.
TITLE    Direct Submission
JOURNAL   Submitted (10-JUL-2000) Yoshihide Hayashizaki, The Institute of
Physical and Chemical Research (RIKEN), Laboratory for Genome
Exploration Research Group, RIKEN Gemomic Sciences Center (GSC),
RIKEN Yokohama Institute; 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama,
Kanagawa 230-0045, Japan (E-mail:genome-res@gsc.riken.go.jp,
URL:

http://genome.gsc.riken.go.jp/

, Tel:81-45-503-9222,
Fax:81-45-503-9216)
COMMENT Please visit our web site (

http://genome.gsc.riken.go.jp/

) for
further details.
cDNA library was prepared and sequenced in Mouse Genome
Encyclopedia Project of Genome Exploration Research Group in Riken
Genomic Sciences Center and Genome Science Laboratory in RIKEN.
Division of Experimental Animal Research in Riken contributed to
prepare mouse tissues. First strand cDNA was primed with a primer
[5' GAGAGAGAGAAGGATCCAAGAGCTCTTTTTTTTTTTTTTTTVN 3'], cDNA was
prepared by using trehalose thermo-activated reverse transcriptase
and subsequently enriched for full-length by cap-trapper. cDNA went
through one round of normalization to Rot = 7.5 and subtraction to
Rot = 37.5. Second strand cDNA was prepared with the primer adapter
of sequence [5'
GAGAGAGAGATTCTCGAGTTAATTAAATTAATCCCCCCCCCCCCC 3']. cDNA was cleaved
with XhoI and SstI. Cloning sites, 5' end: XhoI; 3' end: SstI.
Host: SOLR.
FEATURES    Location/Qualifiers
   source 1..264
   /organism="Mus musculus"
   /strain="C57BL/6J"
   /db_xref="taxon:10090"
   /db_xref="MGD:MGI:102963"
   /db_xref="MGD:MGI:1898920"
   /clone="2810406G07"
   /clone_lib="RIKEN full-length enriched mouse cDNA library"
   /dev_stage="10, 11 days embryo"
   Protein    1..264
   /name="putative"
   CDS    1..264
   /coded_by="AK013010.1:45..839"
ORIGIN
1 megkvyigge dyekeftpkd ylttyysfhs gpvaeqeivk fslqnlyqtf stggvggdvl
   61 idigsgptiy qllsacevfr eiivtdytpq nmqelqkwlk kepgaydwss ivqhaceleg
121 drsrwqekea klrrtvtrvl rcdvtktppl gsaqvpladc vltflameca cpdidtyraa
181 lrrlagllkp gghlvtlvtl rfqhymvgpk kfsgvyleke vvekaiqdag cqvlkcncvs
241 lsyseaycsh dglcfvvark gpsa
//

Begin with the dissolution of superfluous matters
So that desire and consciousness are free

Quicksilver · « **Reply #18 on:** March 24, 2001, 07:47:00 PM »

What is missing is still the enzyme for this step:

N,N-dimethyltryptamine -> psilocin

Try a blast/medline search for that reaction and see what you get.
We already have candidates for the others. (you just dug up similar ones from different species)

you should also consider up-regulating production of S-adenosylmethionine if you are going to express the INMT at high levels, otherwise it might become a limiting factor in the reaction

Isn't this called SAMe? It can be purchased OTC and added as needed, instead of doing some sort of biological manipulation.
-quicksilver-

Teonanacatl · « **Reply #19 on:** April 10, 2001, 12:55:00 PM »

Well...I looked...it ain't there...this isn't surprising, as the thesis on molecular biology of Psilocybe species (which is recent) noted that this step had not been characterized...there are a number of tyrosine monooxygenases and there is a hit for tryptophan-5-monooxygenase...probably the best way to get the sequence for the hydroxylation of DMT (or NMT/T; it's unknown which step happens first in-vivo) would be PCR amplification of a conserved sequence for aromatic monooxyengases, esp. the Trp-5-monooxygenase...I've run a ClustalX protein sequence alignment on phenylalanine hydroxylase, Trp-5-monooxygenase and Tyr-4-monooxygenase and there is surprisingly high similarity between them all...one conserved region that you could recheck w/ a nucleotide sequence alignment is:
IGLASLGA which would come out as 24 bp in nucleotides...unfortunately I don't have any of these in nucleotide form at the moment so I can't calculate the degeneracy that would be present in the PCR primers, but the protein sequence is perfectly conserved here!
Whatcha think?

Begin with the dissolution of superfluous matters
So that desire and consciousness are free

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