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Biochemistry. 2010 Nov 2;49(43):9249-55. doi: 10.1021/bi101291d.

Multiple turnovers of the nicotino-enzyme PdxB require α-keto acids as cosubstrates.

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Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, CO 80309, USA


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 α-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 α-ketoglutarate, oxaloacetic acid, and pyruvate are equally good substrates for PdxB (k(cat)/K(m) values ~1 × 10(4) M⁻¹s⁻¹). The kinetic parameters for the substrate 4PE include a k(cat) of 1.4 s⁻¹, a K(m) of 2.9 μM, and a k(cat)/K(m) of 6.7 × 10(6) M⁻¹s⁻¹. Additionally, we have characterized the stereochemistry of α-ketoglutarate reduction by showing that d-2-HGA, but not l-2-HGA, is a competitive inhibitor vs 4PE and a noncompetitive inhibitor vs α-ketoglutarate.

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