Abstract

DmpFG is bifunctional aldolase/dehydrogenase which catalyzes the last two steps of the meta -cleavage pathway of catechol in Pseudomonas sp. strain CF600. 4-Hydroxy-2-ketovalerate undergoes divalent cation-dependent aldol cleavage to yield pyruvate and acetaldehyde, and the acetaldehyde is then oxidized to acetyl-CoA in an NAD + - and CoA-dependent reaction. Because of its unique primary sequence, structure, and requirement for Mn 2+ , DmpG (aldolase) apparently represents a new sub-class of class II aldolases. X-ray crystallography of DmpFG identified His200 and His202 of a conserved HXH motif in DmpG as potential active-site residues; these histidines were observed to be complexed with a metal ion, which in turn was complexed with a molecule of substrate-analogue (Manjasetty, B. A., Powlowski, J., Vrielink, A. (2003) Proc. Natl. Acad. Sci. 100(12) 6992-6997). To evaluate the structural and functional importance of His200 and His202 of DmpG, H200A, H202A, H203A and H202AH203A variants were constructed, expressed and purified. (Abstract shortened by UMI.)