Contribution to catalysis of ornithine binding residues in ornithine N5-monooxygenase

Abstract

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.

Flavin-dependent N-hydroxylating enzymes: distribution and application

“…Four residues involved in ornithine binding were individually changed to Ala (Robinson et al 2015 ). The Lys107Ala variant lost its hydroxylation activity, indicating that the ionic interaction between Lys107 and the substrate carboxylate is essential for catalysis.…”

Molecular characterization of siderophore biosynthesis in Paracoccidioides brasiliensis

“…SidA is an enzyme specific for hydroxylation of ornithine, a precursor molecule of ferricrome siderophores biosynthesis (Chocklett and Sobrado 2010 ). The three-dimensional structure of SidA in A. fumigatus demonstrates the presence of amino acid residues essential for both NAPDH association and ornithine binding and hydroxylation (Robinson et al 2015 ; Robinson et al 2014 ). In this sense, SidA structure from P. brasiliensis was studied since the capacity of ornithine hydroxylation and use of NADPH as cofactor are specific to this enzyme (Chocklett and Sobrado 2010 ), which are influenced by the amino acids present in its structure (Robinson et al 2014 ; Robinson et al 2015 ).…”

“…The three-dimensional structure of SidA in A. fumigatus demonstrates the presence of amino acid residues essential for both NAPDH association and ornithine binding and hydroxylation (Robinson et al 2015 ; Robinson et al 2014 ). In this sense, SidA structure from P. brasiliensis was studied since the capacity of ornithine hydroxylation and use of NADPH as cofactor are specific to this enzyme (Chocklett and Sobrado 2010 ), which are influenced by the amino acids present in its structure (Robinson et al 2014 ; Robinson et al 2015 ). The characterization of the three-dimensional SidA structure of P. brasiliensis was performed by molecular modeling based on A. fumigatus crystal, which is most similar structure available (Sanchez et al 2017 ).…”

Identification of structural determinants of NAD(P)H selectivity and lysine binding in lysine N-monooxygenase

“…In addition, steady-state kinetics have shown that substrate binding (Km(L-Orn)) was increased ~ 15-fold with no observed effect on NADPH binding. Thus, N323 was proposed to hydrogen bond with the nicotinamide ribose of 17 NADPH as well as contribute to L-Orn binding to balance flavin reduction and substrate binding [14]. In Sida, the interaction of N323 with NADPH was proposed to regulate the position of NADP + , which is believe to require significant movement [22].…”

“…NbtG is a member of the class B flavin monooxygenases, and is grouped under the subfamily of N-hydroxylating monooxygenases (NMOs) [12]. The best characterized NMOs are the L-ornithine (L-Orn) monooxygenases from Aspergillus fumigatus (SidA) and Pseudomonas aeruginosa (PvdA) [13][14][15][16][17][18]. SidA displays an ~ 20-fold preference for NADPH over NADH, while PvdA is only able to react with NADPH [13,19,20].…”