Identification of active site residues involved in substrate binding and cofactor specificity in a putrescine N-monooxygenase

The putrescine N-monooxygenase (NMO) FbsI from Acinetobacter baumannii is a flavin-dependent enzyme that catalyzes the NADPH-dependent hydroxylation of putrescine to N-hydroxyputrescine, an important component of the siderophore fimsbactin A. Here, we probe the roles of T240, D390, and K223 in substrate binding and cofactor recognition. Site-directed mutagenesis and biochemical characterization showed that mutation of T240 to alanine resulted in a >500-fold increase in the K_M for putrescine, with little effect on the k_cat value, highlighting the importance of this residue in binding. Mutation of D390 to alanine and asparagine rendered insoluble or inactive protein, respectively, suggesting this residue is essential for catalysis. Specificity for NAD(P)H was probed by mutating K223 to alanine and arginine. The K223R mutant had a 9-fold lower K_M with NADPH, while K223A had a 2-fold lower k_cat value and minimal change to the K_M value when compared to wild-type (WT) enzyme. However, rapid-reaction kinetics showed that K223R had a >15-fold lower K_D with NADPH while K223A had a 3-fold higher K_D and 7.5-fold lower k_red compared to WT. These results demonstrate that mutation of K223 to arginine increases the specificity and efficiency of the enzyme for NADPH, identifying a key residue in cofactor recognition in FbsI.