Quantitative functional characterization of conserved molecular interactions in the active site of mannitol 2-dehydrogenase.
Enzyme active site residues are often highly conserved, indicating a significant role in function. In this study we quantitate the functional contribution for all conserved molecular interactions occurring within a Michaelis complex for mannitol 2-dehydrogenase derived from Pseudomonas fluorescens (pfMDH). Through systematic mutagenesis of active site residues, we reveal that the molecular interactions in pfMDH mediated by highly conserved residues not directly involved in reaction chemistry can be as important to catalysis as those directly involved in the reaction chemistry. This quantitative analysis of the molecular interactions within the pfMDH active site provides direct insight into the functional role of each molecular interaction, several of which were unexpected based on canonical sequence conservation and structural analyses.
Genome Center, University of California, Davis, California, 95616.
James E
JE
Lucas
Genome Center, University of California, Davis, California, 95616.|Department of Chemistry, University of California, Davis, California, 95616.|Department of Biochemistry & Molecular Medicine, University of California, Davis, California, 95616.
Justin B
JB
Siegel
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics* / Q000502:physiology; D017124:Conserved Sequence; D008354:Mannitol Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011551:Pseudomonas fluorescens / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
D001426:Bacterial Proteins; D008354:Mannitol Dehydrogenases
binding; catalysis; conserved; enzyme; interactions; mannitol 2-dehydrogenase; molecular; mutagenesis; quantitative; specificity