A.1 Ring-hydroxylating dioxygenases
The NAD(P)H-dependent bacterial dioxygenases are multi-component enzyme systems (Cerniglia, 1992), each containing 2 to 4 subunits, depending on the substrate and the source of the enzyme. Each subunit may contain 1 to 2 Fe as the prosthetic group. The subunits serve two main functions: (a) electron transfer from the reducing equivalent NAD(P)H; and (b) hydroxylation or enzymatic. These enzymes are responsible for the initial cis dihydroxylation of an aromatic compound to form a cis-dihydrodiol. This reaction breaks up the aromaticity in the product.
There are three structurally distinct groups of ring-hydroxylating dioxygenases. In the most common group 1, the system is composed of 4 subunits. Subunit I is a flavoprotein reductase. Subunit II is a [2Fe-2S] ferredoxin. These two subunits form the electron transfer component. Subunits & form the catalytic hydroxylase component which is also known as the iron sulfur protein (ISP). The overheads show the organization of various subunits in naphthalene dioxygenase (from Cerniglia, 1992) and toluene dioxygenase (Zylstra & Gibson, 1991). The multicomponent enzyme system of naphthalene dioxygenase is very similar to that of benzene or toluene dioxygenases. There is an electron transfer between these proteins, resulting in movement of the reducing equivalents from NAD(P)H to the terminal dioxygenase.
Group 1 ring-hydroxylating dioxygenases are represented by benzene dioxygenase, toluene dioxygenase, biphenyl dioxygenase and naphthalene dioxygenase. It is believed that they also attack the higher polycylic aromatic hydrocarbons (PAHs) such as phenanthrene, anthracene and benz[a]anthracene, etc. The transformation of some of these compounds into their corresponding cis-dihydrodiols is shown in the overhead. Note that except for benzene which does not possess any other substituent groups on the aromatic ring, hydroxylation occurs right next to a carbon substituent group attached to the ring in all cases. Again, there are exceptions. For example, biphenyl dioxygenases can hydroxylate at the 3,4- positions.
Group 2 ring-hydroxylating dioxygenases are represented by benzoate and toluate dioxygenases, each containing 3 subunits. Subunit 1 mediates electron transfer from NAD(P)H to the hydroxylase, while subunits & form the catalytic hydroxylase ISP component. In reactions of this type (eg. with benzoic acid), one hydroxyl group goes to the carbon bearing the carboxylate substituent, while the other goes to the adjacent carbon, ie. positions 1 & 2 are being hydroxylated. This enzyme can accommodate other substituent groups such as sulphonate, (-SO3) or nitro (-NO2) groups.
Group 3 ring-hydroxylating dioxygenases are typified by phthalate dioxygenase which contains 2 subunits. One serves electron transfer function while the other serves the hydroxylase function. This enzyme hydroxylates at positions 4 & 5 of the phthalate ring, and this pattern is very different from the other hydroxylating dioxygenases.
The substrate specificity of ring-hydroxylating dioxygenases is determined by the terminal iron-sulfur proteins or hydroxylases. This was shown in the elegant studies of Furukawa's group using hybrid toluene/biphenyl dioxygenases by mixing components from toluene dioxygenase of Pseudomonas putida F1 and from biphenyl dioxygenase of Pseudomonas pseudoalcaligenes KF707 (described in Ensley, 1994). Replacing both the small and large subunits of the biphenyl dioxygenase with those of toluene hydroxylase led to a hybrid enzyme with specificity for toluene. Replacing only the large subunit of the biphenyl dioxygenase with that of toluene dioxygenase led to a hybrid enzyme with specificity for both biphenyl and toluene. Furthermore, both types of hybrid enzymes now exhibit the ability to oxidize trichloroethylene (TCE). This latter finding was completely unexpected, but a pleasant surprise, because TCE is one of the most important pollutants in groundwater.
Dihydrodiol dehydrogenases
cis-Dihydrodiols produced from ring-hydroxylating dioxygenases are converted by dihydrodiol dehydrogenases to catechols. This step removes 2 H's from the cis-dihydrodiol to restore the aromatic nature of the parent ring and produce a dihydroxy derivative. The reaction has a requirement for NAD, ie. the NADH used in the hydroxylating reaction is regenerated. Now, the product is ready for the ring-cleaving type of dioxygenase attack. Several dihydrodiol dehydrogenases have been characterized. Most are composed of tetramers of identical subunits. They are members of the short-chain-alcohol/polyol dehydrogenase superfamily of enzymes which differ from the zinc-containing alcohol dehydrogenases. Note that ortho-substituted products derived from group 2 hydroxylating dioxygenases (eg. from benzoate dioxgenase) are generally unstable, and they transform spontaneously into catechol. Therefore, there is no regeneration of NADH for compounds taking this metabolic route.
Well, there you have it. Proof. Now I need to dig up another ref. citation for a biphenyl dioxygenase. The point is that a (3,4- methylenedioxy) group IS obtainable and can be added to the a substitued aromatic ring.
Flip
Conclusion /nm./: the place where you got tired of thinking.
