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Primary and Solvent Kinetic Isotope Effects on Catalysis by Aspergillus fumigatus Ornithine Hydroxylase
Author(s) -
Romero Elvira,
Fedkenheuer Mike,
Sobrado Pablo
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.737.4
Subject(s) - chemistry , kinetic isotope effect , flavin group , aspergillus fumigatus , hydroxylation , reductase , stereochemistry , enzyme kinetics , catalysis , ornithine , medicinal chemistry , photochemistry , active site , enzyme , organic chemistry , biochemistry , deuterium , arginine , amino acid , physics , quantum mechanics , microbiology and biotechnology , biology
Aspergillus fumigatus siderophore A (AfSidA) is a flavin containing monooxygenase that catalyzes the NADPH‐dependent hydroxylation of the side chain amino group of ornithine in the biosynthesis of ferricrocin. Mutant strains of A. fumigatus having a deletion of the AfSidA gene are avirulent in mouse models, suggesting that this enzyme is a potential drug target. The AfSidA reaction consists of the oxidative and reductive half‐reactions, where the flavin is reduced by NADPH followed by reaction with oxygen leading to the formation of an oxygenated flavin intermediate. Kinetic isotope effects with deuterated pyrimidines were measured to probe the reductive half‐reaction. With NADPD, a D k cat value of 3.2 ± 0.2 and a D k cat /K M value of 2.7 ± 0.2 were measured. Using NADD, the D k cat and D k cat /K M values were 2.0 ± 0.1 and 2.5 ± 0.3, respectively. The oxidative half‐reaction was probed by solvent isotope effects experiments. Using NADPH as the reductant, a D2O k cat value of 1.5 ± 0.2 and a D2O (k cat /K M ) value of 1.4 ± 0.1 were measured. Using NADH the D2O k cat value was 1.7 ± 0.2 and the D2O k cat /K M value was 1.0 ± 0.1. These results clearly show that hydride transfer and a proton transfer steps are partially rate limiting in the reaction of AfSidA. The differences in the magnitude of the isotope effects between the pyrimidines and the origin of the solvent kinetic isotope effect will be discussed. This research was funded by a grant from NSF (MCB‐1021384)