Multiple Turnovers of the Nicotino-Enzyme PdxB Require α-Keto Acids as Cosubstrates

PdxB catalyzes the second step in the biosynthesis of pyridoxal phosphate by oxidizing 4-phospho-D-erythronate (4PE) to 2-oxo-3-hydroxy-4-phosphobutanoate (OHPB) with concomitant reduction of NAD(+) to NADH. PdxB is a nicotino-enzyme wherein the NAD(H) cofactor remains tightly bound to PdxB. It has been a mystery how PdxB performs multiple turnovers since addition of free NAD(+) does not reoxidize the enzyme-bound NADH following conversion of 4PE to OHPB. We have solved this mystery by demonstrating that a variety of physiologically available alpha-keto acids serve as oxidants of PdxB to sustain multiple turnovers. In a coupled assay using the next two enzymes of the biosynthetic pathway for pyridoxal phosphate (SerC and PdxA), we have found that alpha-ketoglutarate, oxaloacetic acid, and pyruvate are equally good substrates for PdxB (k(cat)/K-m values similar to 1 x 10(4) M-1 s(-1)). The kinetic parameters for the substrate 4PE include a k(cat) of 1.4 s(-1), a K-m of 2.9 mu M, and a k(cat)/K-m of 6.7 x 10(6) M-1 s(-1). Additionally, we have characterized the stereochemistry of alpha-ketoglutarate reduction by showing that D-2-HGA, but not L-2-HGA, is a competitive inhibitor vs 4PE and a noncompetitive inhibitor vs alpha-ketoglutarate.