5cku
Structure of Aspergillus fumigatus ornithine hydroxylase (SidA) mutant N323A bound to NADP and ornithine
Structural highlights
FunctionSIDA_ASPFU Catalyzes the conversion of L-ornithine to N(5)-hydroxyornithine, the first step in the biosynthesis of all hydroxamate-containing siderophores, such as the secreted triacetylfusarinine C (TAFC) involved in iron uptake and the intracellular iron storage compound desferriferricrocin (DFFC). Highly specific for its substrate, only hydrolyzing l-ornithine. Has preference for NADPH over NADH, NADPH playing a role in stabilization of the C4a-hydroperoxyflavin intermediate. Essential for virulence.[1] [2] [3] [4] Publication Abstract from PubMedThe SidA ornithine N5-monooxygenase from Aspergillusfumigatus 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. Contribution to catalysis of ornithine binding residues in ornithine N5-monooxygenase.,Robinson R, Qureshi IA, Klancher CA, Rodriguez PJ, Tanner JJ, Sobrado P Arch Biochem Biophys. 2015 Sep 12;585:25-31. doi: 10.1016/j.abb.2015.09.008. PMID:26375201[5] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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