(a) Tetrameric assembly of the bifunctional enzyme DmpFG. The two DmpG molecules are shown in different shades of blue and the two DmpF protomers, located at the peripheral ends of the oligomer, are represented in different shades of red. The Mn²⁺ bound to DmpG is shown in rust and the oxalate and NAD⁺ bound to the active sites of DmpG and DmpF, respectively, are shown as ball-and-stick representations. (b) Stereo representation of the DmpG protomer. The TIM barrel domain is magenta and the helical communication domain is green. The helices that have been inserted into the barrel domain are shown in yellow. (c) Stereoview of the secondary structure of DmpF. The NAD⁺ domain is shown in red and the dimerization domain is in blue. (d) Worm representation showing the superposition of the holo form of DmpF (yellow) and the apo form of DmpF (purple). The α carbon atoms from the dimerization domains of DmpF (residues 131–285) for the holo and apo structures were used for the superposition. The NAD⁺ cofactor bound to the apoenzyme in c and d is represented as a ball-and-stick model.

(a and b) Worm representation of DmpFG showing the apo form with the buried surface of the intermediate channel (a) and the holo form with the buried surface of the intermediate channel extending completely between the two active sites (b). (c and d) Worm representation showing a close-up view of the buried surface of the intermediate channel around the second barrier for the apo form (c) and the holo form (d) of the enzyme. The side chains of the residues that adopt multiple conformations at this barrier point are shown in a single conformation. In the holo form (d) this conformational arrangement results in an opening of the second barrier point from the intermediate tunnel to the active site of the DmpF molecule. (e and f) Worm representation showing the substrate entrance tunnel in the closed conformation blocked by the side chain of His-21 (e) and the open substrate entrance channel with His-21 in an alternative side-chain conformation (f). The DmpG chain is shown in purple and the DmpF chain is in magenta. Specific amino acid residues and the NAD cofactor are represented as ball-and-stick models, and the Mn²⁺ cofactor is shown as a yellow sphere. The intermediate channel is blue and the substrate entrance channel is gray. Hydrogen bonding interactions are shown in dotted lines. The solvent-accessible surfaces were computed with the program spock (23), using a probe radius of 1.4 Å.

A bifunctional aldolase–dehydrogenase catalyzes the final two steps of the meta-cleavage pathway for catechol, an intermediate in many bacterial species in the degradation of phenols, toluates, naphthalene, biphenyls and other aromatic compounds (reviewed in ref. 12). Thus, 4-hydroxy-2-ketovalerate aldolase (DmpG; EC 4.1.3.-) and acetaldehyde dehydrogenase (acylating) (DmpF; EC 1.2.1.10) from a methylphenol-degrading pseudomonad convert 4-hydroxy-2-ketovalerate to pyruvate and acetyl-CoA by way of the intermediate acetaldehyde (Scheme 1).

(a) Active site of DmpG. The Mn²⁺ ion is shown as a yellow sphere. Red spheres correspond to bound water molecules. The oxalate ligand is shown with blue bonds. The dotted lines represent coordinating interactions between active site residues and the bound metal ion. (b)2F_o – F_c electron density in the active site of DmpG. The density is contoured at a 1.5-σ level. (c) Model of the substrate-bound complex of DmpG. The modeled substrate, 4-hydroxy-2-ketovalerate, is shown in blue bonds. The secondary structure elements are colored as described for Fig. 1.

(a–e) Schematic representation of the postulated reaction mechanism for DmpG and the opening of the intermediate channel by Tyr-291. (f) Model of the Tyr-291 residue in the alternative conformation resulting in the channel open state. The protein chains are shown in worm representation with DmpG blue and DmpF magenta.