Lys-D48 is required for charge stabilization, rapid flavin reduction, and internal electron transfer in the catalytic cycle of dihydroorotate dehydrogenase B of Lactococcus lactis.

The Journal of Biological Chemistry
Jonathan P CombeNigel S Scrutton

Abstract

Dihydroorotate dehydrogenase B (DHODB) catalyzes the oxidation of dihydroorotate (DHO) to orotate and is found in the pyrimidine biosynthetic pathway. The Lactococcus lactis enzyme is a dimer of heterodimers containing FMN, FAD, and a 2Fe-2S center. Lys-D48 is found in the catalytic subunit and its side-chain adopts different positions, influenced by ligand binding. Based on crystal structures of DHODB in the presence and absence of orotate, we hypothesized that Lys-D48 has a role in facilitating electron transfer in DHODB, specifically in stabilizing negative charge in the reduced FMN isoalloxazine ring. We show that mutagenesis of Lys-D48 to an alanine, arginine, glutamine, or glutamate residue (mutants K38A, K48R, K48Q, and K48E) impairs catalytic turnover substantially (approximately 50-500-fold reduction in turnover number). Stopped-flow studies demonstrate that loss of catalytic activity is attributed to poor rates of FMN reduction by substrate. Mutation also impairs electron transfer from the 2Fe-2S center to FMN. Addition of methylamine leads to partial rescue of flavin reduction activity. Nicotinamide coenzyme oxidation and reduction at the distal FAD site is unaffected by the mutations. Formation of the spin-interacti...Continue Reading

References

May 7, 2003·The Journal of Biological Chemistry·Sofie NøragerSine Larsen
Mar 27, 2004·Protein Science : a Publication of the Protein Society·Majbritt HansenSine Larsen

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