Contribution to catalysis of ornithine binding residues in ornithine N5-monooxygenase.
Publication Type:Journal Article
Source:Arch Biochem Biophys, Volume 585, p.25-31 (2015)
Keywords:Aspergillus fumigatus, Biocatalysis, Crystallography, X-Ray, Escherichia coli, Flavins, Fungal Proteins, Gene Expression, Hydrogen Bonding, Hydroxylation, Kinetics, Mixed Function Oxygenases, Models, Molecular, Mutagenesis, Site-Directed, NADP, Ornithine, Oxidation-Reduction, Recombinant Proteins, Structure-Activity Relationship, Substrate Specificity
<p>The SidA ornithine N5-monooxygenase from Aspergillus fumigatus is a flavin monooxygenase that catalyzes the NADPH-dependent hydroxylation of ornithine. Herein we report a mutagenesis study targeting four residues that contact ornithine in crystal structures of SidA: Lys107, Asn293, Asn323, and Ser469. Mutation of Lys107 to Ala abolishes activity as measured in steady-state oxygen consumption and ornithine hydroxylation assays, indicating that the ionic interaction of Lys107 with the carboxylate of ornithine is essential for catalysis. Mutation of Asn293, Asn323, or Ser469 individually to Ala results in >14-fold increases in Km values for ornithine. Asn323 to Ala also increases the rate constant for flavin reduction by NADPH by 18-fold. Asn323 is unique among the four ornithine binding residues in that it also interacts with NADPH by forming a hydrogen bond with the nicotinamide ribose. The crystal structure of N323A complexed with NADP(+) and ornithine shows that the nicontinamide riboside group of NADP is disordered. This result suggests that the increase in flavin reduction rate results from an increase in conformational space available to the enzyme-bound NADP(H). Asn323 thus facilitates ornithine binding at the expense of hindering flavin reduction, which demonstrates the delicate balance that exists within protein-ligand interaction networks in enzyme active sites.</p>