Kinetic and Structural Characterization of a Flavin-Dependent Putrescine N-Hydroxylase from Acinetobacter baumannii

Abstract

Acinetobacter baumannii is a Gram-negative opportunistic pathogen that causes nosocomial infections, especially among immunocompromised individuals. The rise of multidrug resistant strains of A. baumannii has limited the use of standard antibiotics, highlighting a need for new drugs that exploit novel mechanisms of pathogenicity. Disrupting iron acquisition by inhibiting the biosynthesis of iron-chelating molecules (siderophores) secreted by the pathogen is a potential strategy for developing new antibiotics. Here we investigated FbsI, an N-hydroxylating monooxygenase involved in the biosynthesis of fimsbactin A, the major siderophore produced by A. baumannii. FbsI was characterized using steady-state and transient-state kinetics, spectroscopy, X-ray crystallography, and small-angle X-ray scattering. FbsI was found to catalyze the N-hydroxylation of the aliphatic diamines putrescine and cadaverine. Maximum coupling of the reductive and oxidative half-reactions occurs with putrescine, suggesting it is the preferred (in vivo) substrate. FbsI uses both NADPH and NADH as the reducing cofactor with a slight preference for NADPH. The crystal structure of FbsI complexed with NADP+ was determined at 2.2 Å resolution. The structure exhibits the protein fold characteristic of Class B flavin-dependent monooxygenases. FbsI is most similar in 3D structure to the cadaverine N-hydroxylases DesB and DfoA. Small-angle X-ray scattering shows that FbsI forms a tetramer in solution like the N-hydroxylating monooxygenases of the SidA/IucD/PvdA family. A model of putrescine docked into the active site provides insight into substrate recognition. A mechanism for the catalytic cycle is proposed where dehydration of the C4a-hydroxyflavin intermediate is partially rate-limiting, and the hydroxylated putrescine product is released before NADP+.

A biosynthetic aspartate N-hydroxylase performs successive oxidations by holding intermediates at a site away from the catalytic center

“…The enzyme is a monomer comprising 22 α-helices and 17 β-strands arranged in a three-domain architecture ( Fig. 5 A ) ( 16 , 17 , 32 ). The active site is in a cleft at the interface of the three domains ( Fig.…”

Kinetic Characterization and Identification of Key Active Site Residues of the L‐Aspartate N‐Hydroxylase, CreE

“…k OH was the slowest step that was observed in the catalytic cycle, yet it is ~2-fold faster than the k cat value measured for CreE (Tables 1 & 3). [38] This was also observed for FzmM, where k OH was ~1.6-fold faster than the k cat value. [17] Because CreE (and FzmM) catalyzes three rounds of oxidation, it is possible that flavin dehydration of the second or third oxidation step becomes slower and could correspond to the observed k cat value.…”

“…The data showed a minimal effect on the k cat , with increases in the K M value, which is consistent with NADP + acting as a competitive inhibitor (Figure S2). [17,38] A K I value for NADP + of 0.31 � 0.07 mM was calculated (Figure S2).…”

“…In the structure of SV2À NMO, NADP + is bound in the same conformation and location as that observed in SidA and other NMOs. [31,38,44] Thus, we considered that residues that were positively charged and/or could participate in hydrogen bonding within ~10 Å from the NADP + ribose 2'-OH and the pyrimidine ring of flavin could be involved in binding Laspartate (Figure S5). Using these parameters, T65, R291, R440, and R443 were selected.…”

Unifying and versatile features of flavin-dependent monooxygenases: Diverse catalysis by a common C4a-(hydro)peroxyflavin

“…Among these, enzymes catalyzing the hydroxylation of nitrogen known as NMOs have recently been investigated extensively for their structural and mechanistic properties. NMOs belong to the single-component group B of FDMO ( 65 , 66 , 67 , 68 ). The enzymes catalyze N-hydroxylation reactions of amine-containing compounds and use NAD(P)H as their reductant with a preference for NADPH over NADH ( Fig.…”