Identification of Active Site Residues Involved in Substrate Binding and Cofactor Specificity in a Putrescine N-monooxygenase

Abstract

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 KM for putrescine, with little effect on the kcat 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 KM with NADPH, while K223A had a 2-fold lower kcat value and minimal change to the KM value when compared to wild-type (WT) enzyme. However, rapid-reaction kinetics showed that K223R had a >15-fold lower KD with NADPH while K223A had a 3-fold higher KD and 7.5-fold lower kred 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.

Department(s)

Chemistry

Comments

Life Sciences Institute, Grant CHE-2003658

Keywords and Phrases

Flavin-dependent monooxygenases; Mutagenesis; N-hydroxylating monooxygenases; putrescine; Steady-state kinetics; Stopped-flow spectroscopy

International Standard Serial Number (ISSN)

1096-0384; 0003-9861

Document Type

Article - Journal

Document Version

Citation

File Type

text

Language(s)

English

Rights

© 2025 Elsevier, All rights reserved.

Publication Date

01 Sep 2025

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