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

Abstract

The soil actinomycete Kutzneria sp. 744 produces a class of highly decorated hexadepsipeptides, which represent a new chemical scaffold that has both antimicrobial and antifungal properties. These natural products, known as kutznerides, are created via nonribosomal peptide synthesis using various derivatized amino acids. The piperazic acid moiety contained in the kutzneride scaffold, which is vital for its antibiotic activity, has been shown to derive from the hydroxylated product of l-ornithine, l-N5-hydroxyornithine. The production of this hydroxylated species is catalyzed by the action of an FAD- and NAD(P)H-dependent N-hydroxylase known as KtzI. We have been able to structurally characterize KtzI in several states along its catalytic trajectory, and by pairing these snapshots with the biochemical and structural data already available for this enzyme class, we propose a structurally based reaction mechanism that includes novel conformational changes of both the protein backbone and the flavin cofactor. Further, we were able to recapitulate these conformational changes in the protein crystal, displaying their chemical competence. Our series of structures, with corroborating biochemical and spectroscopic data collected by us and others, affords mechanistic insight into this relatively new class of flavin-dependent hydroxylases and adds another layer to the complexity of flavoenzymes.

Structural and mechanistic basis of differentiated inhibitors of the acute pancreatitis target kynurenine-3-monooxygenase

“…Many of these residues lie within the solvent channel identified as accessible from the active site in our tilting flavin structures, enabling us to model a NADPH molecule along this channel to place the nicotinamide ring on the re face of the flavin. Interestingly, this ring position closely resembles that found in the catalytically competent FAD-NADPH-substrate complexes of class B monoxygenases283233 (Fig. 7c,d, Supplementary Fig.…”

“…A range of flavin conformations has been captured by structural studies, leading to terms such as ‘waving'252627 and ‘flapping'28 flavin to describe the repertoire of motions observed. The tilting flavin orientation seen for series B and C inhibitors does not match any of those reported and is much more appropriately described as a tilt, as it lacks the lateral motion for the wave that leads to the ‘out' state first reported in the class A exemplar enzyme PHBH (Fig.…”

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

“…The four NbtG molecules present in the asymmetric unit form a compact tetramer with the same arrangement observed in the L-Orn monooxygenases SidA (10) and PvdA (11) (their monomeric and dimeric structures, respectively, form a tetramer through application of crystallographic symmetry operators); in KtzI a similar but not identical tetramer was observed (12). The NbtG subunit folds into a two-domain topology similar to that of other NMOs: the FAD binding domain (residues 1-178 and 337-429), including the non-covalently bound FAD cofactor (Fig.…”

Contribution to catalysis of ornithine binding residues in ornithine N5-monooxygenase

“…The biosynthesis of HCS in A. fumigatus (i.e., triacetylfusarine and hydroxyferricrocin) is initiated by the hydroxylation of ornithine by SidA, a member of the flavin-dependent Class B monooxygenases [6, 41]. The binding site of Orn in SidA is conserved among the other Orn monooxygenases, PvdA and KtzI [22, 37]. The binding site provides these enzymes with specificity for Orn.…”

“…These residues are conserved in related Orn monooxygenases from Pseudomona aeruginosa , PvdA, and from Kutzneria sp. 744, KtzI [22, 23]. These four residues were mutated to Ala in order to determine their effect on Orn binding and role in catalysis.…”