Structural and Kinetic Properties of a beta-Hydroxyacid Dehydrogenase Involved in Nicotinate Fermentation.

Abstract 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 β-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°. 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.