title: 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.
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-interacting state between the FMN semiquinone-reduced 2Fe-2S centers observed in wild-type enzyme is retained in the mutant proteins, consistent with there being little perturbation of the superexchange paths that contribute to the efficiency of electron transfer between these cofactors. Our data suggest a key charge-stabilizing role for Lys-D48 during reduction of FMN by dihydroorotate, or by electron transfer from the 2Fe-2S center, and establish a common mechanism of FMN reduction in the single FMN-containing A-type and the complex multicenter B-type DHOD enzymes.
authors.affiliation: Faculty of Life Sciences, Manchester Interdisciplinary Biocentre, University of Manchester, Jackson's Mill, Sackville Street, Manchester M60 1QD, United Kingdom.
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: Combe
authors.forename: Jaswir
authors.initials: J
authors.lastname: Basran
authors.forename: Parvinder
authors.initials: P
authors.lastname: Hothi
authors.forename: David
authors.initials: D
authors.lastname: Leys
authors.forename: Stephen E J
authors.initials: SE
authors.lastname: Rigby
authors.forename: Andrew W
authors.initials: AW
authors.lastname: Munro
authors.forename: Nigel S
authors.initials: NS
authors.lastname: Scrutton
mesh_terms: D002384:Catalysis; D000090722:Dihydroorotate Dehydrogenase; D019281:Dimerization; D004563:Electrochemistry; D004579:Electron Transport / Q000502:physiology*; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism*; D005415:Flavins / Q000737:chemistry / Q000378:metabolism; D013294:Lactococcus lactis / Q000201:enzymology*; D008239:Lysine / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D009963:Orotic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D010084:Oxidation-Reduction; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D014443:Tyrosine / Q000378:metabolism
chemical_list: D000090722:Dihydroorotate Dehydrogenase; D005415:Flavins; D009243:NAD; C004768:4,5-dihydroorotic acid; D014443:Tyrosine; C008173:isoalloxazine; D009963:Orotic Acid; D005486:Flavin Mononucleotide; D044925:Oxidoreductases Acting on CH-CH Group Donors; D008239:Lysine