Mechanistic and structural studies of the N-hydroxylating flavoprotein monooxygenases

Abstract

The N-hydroxylating flavoprotein monooxygenases are siderophore biosynthetic enzymes that catalyze the hydroxylation of the sidechain amino-group of ornithine or lysine or the primary amino-group of putrescine. This hydroxylated product is subsequently formylated or acylated and incorporated into the siderophore. Importantly, the modified amino-group is a hydroxamate and serves as an iron chelating moiety in the siderophore. This review describes recent work to characterize the ornithine hydroxylases from Pseudomonas aeruginosa (PvdA) and Aspergillus fumigatus (SidA) and the lysine hydroxylase from Escherichia coli (IucD). This includes summaries of steady and transient state kinetic data for all three enzymes and the X-ray crystallographic structure of PvdA.

Role of Ser-257 in the Sliding Mechanism of NADP(H) in the Reaction Catalyzed by the Aspergillus fumigatus Flavin-dependent Ornithine N5-Monooxygenase SidA

“…In the absence of this interaction, NADP ϩ becomes more mobile and unable to acquire the correct position for optimal catalysis. A serine residue homologous to Ser-257 is conserved in all ornithine hydroxylases, including the prokaryotic PvdA (33,37). In addition, a hydroxyl-containing amino acid is conserved in the same position of all known structures of Class B flavin-dependent monooxygenases (Fig.…”

Heteroatom–Heteroatom Bond Formation in Natural Product Biosynthesis

“…This initial reduction ( 13 → 14 ) occurs independently of substrate binding. 63–65 The NMOs EtcB, PvdA, SidA, and CchB are specific for NADPH over NADH in this reduction reaction. 45,48,54,66,67 Next, oxygen is proposed to diffuse from the bulk solvent into the enzyme active site housing the flavin cofactor through enzyme multichannels.…”

“…63–65 The proton source for 16 is proposed to derive from the charged amino acid side chain, as evidenced by a spectroscopic shift from 361nm, which is indicative of a peroxyflavin intermediate, to the 380nm of 16 . 63,65,69 A hallmark of these class B NMOs is the stability of 16 , which has a half-life of about 30 minutes.…”

An Unprecedented NADPH Domain Conformation in Lysine Monooxygenase NbtG Provides Insights into Uncoupling of Oxygen Consumption from Substrate Hydroxylation

“…A different strategy for oxygen consumption regulation was found in KtzI where flavin movement from an "out" to "in" conformation controls the oxidized/reduced state of the cofactor, similarly to what happens in Class A monooxygenases such as p-hydroxybenzoate hydroxylase. Despite this difference, the structures of SidA, PvdA and KtzI all share a similar overall fold to other Class B flavin monooxygenases and a similar binding conformation for NADP ϩ , which is important for stabilization of the FAD OOH (6,10,17,18).…”

“…These enzymes catalyze the NADPH-and oxygen-dependent hydroxylation of amine groups on the side chains of L-ornithine (L-Orn), L-lysine (L-Lys), and some primary aliphatic diamines (1)(2)(3)(4)(5)(6). Many of these NMOs are linked to virulence in pathogenic bacteria.…”