Two Structures of an N-Hydroxylating Flavoprotein Monooxygenase

Abstract

The ornithine hydroxylase from Pseudomonas aeruginosa (PvdA) catalyzes the FAD-dependent hydroxylation of the side chain amine of ornithine, which is subsequently formylated to generate the iron-chelating hydroxamates of the siderophore pyoverdin. PvdA belongs to the class B flavoprotein monooxygenases, which catalyze the oxidation of substrates using NADPH as the electron donor and molecular oxygen. Class B enzymes include the well studied flavin-containing monooxygenases and Baeyer-Villiger monooxygenases. The first two structures of a class B N-hydroxylating monooxygenase were determined with FAD in oxidized (1.9 Å resolution) and reduced (3.03 Å resolution) states. PvdA has the two expected Rossmann-like dinucleotide-binding domains for FAD and NADPH and also a substrate-binding domain, with the active site at the interface between the three domains. The structures have NADP(H) and (hydroxy)ornithine bound in a solvent-exposed active site, providing structural evidence for substrate and cosubstrate specificity and the inability of PvdA to bind FAD tightly. Structural and biochemical evidence indicates that NADP ؉ remains bound throughout the oxidative half-reaction, which is proposed to shelter the flavin intermediates from solvent and thereby prevent uncoupling of NADPH oxidation from hydroxylated product formation.The ornithine hydroxylase from Pseudomonas aeruginosa (PvdA) catalyzes the FAD-dependent hydroxylation of the ornithine side chain amine using NADPH as the electron donor and molecular oxygen (1). As a microbial N-hydroxylating monooxygenase, PvdA is considered to be a member of the class B flavoprotein monooxygenases, which share the following characteristics. 1) FAD is a stably bound cofactor; 2) NADPH, but not NADH, serves as the electron-donating cosubstrate and remains bound during the oxidative half-reaction; 3) they are composed of FAD and NADPH dinucleotidebinding domains; and 4) they are encoded by a single gene (2). PvdA meets these requirements with some caveats: PvdA does not stably bind FAD (1), and no previous work documents structure or confirms NADP ϩ binding through the oxidative half-reaction.The known class B flavoprotein monooxygenases are divided into three subclasses (2). Microbial N-hydroxylating monooxygenases catalyze the hydroxylation of primary amines and include PvdA (1, 3, 4), the ornithine hydroxylase from Aspergillus fumigatus (SidA) (5, 6), and the lysine hydroxylases (7,8). A mechanism for PvdA (Fig. 1a) has been proposed previously (4). In short, NADPH reduces the oxidized flavin in the reductive half-reaction. In the oxidative half-reaction, ornithine binding accelerates the addition of oxygen to the flavin and makes a short-lived peroxyflavin intermediate (which is slow to form and long-lived in the absence of ornithine), followed by the hydroperoxyflavin intermediate. The hydroperoxyflavin donates the distal oxygen atom to the ornithine, forming hydroxyornithine and the hydroxyflavin intermediate. The hydroxyflavin intermediate dehydrates to regenerate the oxi...

Crystallographic Evidence of Drastic Conformational Changes in the Active Site of a Flavin-Dependent N-Hydroxylase

“…The anaerobically reduced KtzI–FADred–NADP + – l -Orn structure (Figure 3b,e) and an accompanying depiction in which a bromide ion from crystallization has displaced the substrate [KtzI–FADred–NADP + –Br (Figure S5a of the Supporting Information)] are very similar to the aerobically reduced depictions of PvdA 30 (Figure S5b of the Supporting Information) and SidA 29 (Figure S5c of the Supporting Information). This similarity indicates that reconstituting the enzyme in the absence of oxygen, as was done for KtzI, can be approximated by chemical reduction of the aerobic state, and thus, these structures can all be taken together as independent validations of the preturnover state of enzymes in this family.…”

“…29,30 In the biochemical studies, PvdA and SidA were proposed to follow a similar reaction mechanism as outlined in Figure 2. Common to most flavin monooxygenases, the oxidized FAD cofactor is reduced by hydride transfer from the C4- pro - R position of the NADPH nicotinamide to the N 5 position of the flavin isoalloxazine ring, completing what is known as the reductive half of the reaction (Figure 2, 1 → 3 ).…”

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

“…Thus, NADPH reduces the flavin, whereas NADP ϩ plays an essential role in the stabilization of flavin intermediates in SidA. This dual or moonlighting role of NADP(H) has also been observed in the reaction of the related ornithine hydroxylase PvdA from Pseudomonas aeruginosa (31)(32)(33). A moonlighting role for NADP(H) was originally proposed for other members of the Class B flavin-dependent monooxygenases (12,15,34).…”

“…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.…”

Substrate Trapping in the Siderophore Tailoring Enzyme PvdQ

“…Structures of pyoverdine biosynthetic enzymes with amino acid precursors have been reported ( e.g. , PvdA 19 ), but to our knowledge, this is the first structure of a pyoverdine precursor peptide in complex with its biosynthetic enzyme. Elucidation of such bioprecursor:enzyme complexes offers an informative contrast with structures of completed siderophores.…”