From malate dehydrogenase to phenyllactate dehydrogenase. Incorporation of unnatural amino acids to generate an improved enzyme-catalyzed activity.
Malate dehydrogenase (MDH) from Escherichia coli is highly specific for its keto acid substrate. The placement of the active site-binding groups in MDH effectively discriminates against both the shorter and the longer keto dicarboxylic acids that could potentially serve as alternative substrates. A notable exception to this specificity is the alternative substrate phenylpyruvate. This aromatic keto acid can be reduced by MDH, albeit at a somewhat slower rate and with greatly diminished affinity, despite the presence of several substrate-binding arginyl residues and the absence of a hydrophobic pocket in the active site. The specificity of MDH for phenylpyruvate has now been enhanced, and that for the physiological substrate oxaloacetate has been diminished, through the replacement of one of the binding arginyl residues with several unnatural alkyl and aryl amino acid analogs. This approach, called site-specific modulation, incorporates systematic structural variations at a site of interest. Molecular modeling studies have suggested a structural basis for the affinity of native MDH for phenylpyruvate and a rationale for the improved catalytic activity that is observed with these new, modified phenyllactate dehydrogenases.
Wright
S K
SK
Department of Chemistry, University of Akron, Akron, Ohio 44325, USA.
Kish
M M
MM
Viola
R E
RE
D000478:Alkylation; D019943:Amino Acid Substitution; D001120:Arginine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D007773:Lactates / Q000378:metabolism*; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D008293:Malates / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010071:Oxaloacetates / Q000378:metabolism; D015202:Protein Engineering; D013379:Substrate Specificity
D007773:Lactates; D008293:Malates; D010071:Oxaloacetates; C017648:3-phenyllactic acid; C030298:malic acid; D001120:Arginine; D008291:Malate Dehydrogenase