Structural and kinetic properties of a beta-hydroxyacid dehydrogenase involved in nicotinate fermentation.
2-(Hydroxymethyl)glutarate dehydrogenase, the fourth enzyme of the anaerobic nicotinate fermentation pathway of Eubacterium barkeri, catalyzes the NADH-dependent conversion between (S)-2-formylglutarate and (S)-2-(hydroxymethyl)glutarate. As shown by its 2.3-A crystal structure, this enzyme is a novel member of the beta-hydroxyacid dehydrogenase family and adopts a tetrameric architecture with monomers interacting via their C-terminal catalytic domains. The NAD-binding domains protrude heterogeneously from the central, tetrameric core with domain rotation angles differing up to 12 degrees. Kinetic properties of the enzyme, including NADH inhibition constants, were determined. A strong NADH binding in contrast to weaker NAD(+) binding of the protein was inferred from fluorometrically determined binding constants for the dinucleotide cofactor. The data support either an Iso Ordered Bi Bi mechanism or a more common Ordered Bi Bi mechanism as found in other dehydrogenases.
Reitz
Simon
S
Philipps-Universität Marburg, Fachbereich Chemie, Hans-Meerwein-Strasse, D-35032 Marburg, Germany.
Alhapel
Ashraf
A
Essen
Lars-Oliver
LO
Pierik
Antonio J
AJ
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D005051:Eubacterium / Q000378:metabolism; D005285:Fermentation; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D009525:Niacin / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D016415:Sequence Alignment
D001426:Bacterial Proteins; D009243:NAD; D009525:Niacin; D002237:Carbohydrate Dehydrogenases; C020843:gulonate dehydrogenase