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...

Edited by R. J. Read, University of Cambridge, England Keywords: pyruvate kinase; allosterism; human liver isozyme. PDB references: human liver pyruvate kinase, D499N variant, 6nn4; W527H variant, 6nn5; Á529/S531G variant, 6nn7; S531E variant, 6nn8Supporting information: this article has supporting information at journals.iucr.org/f Human liver pyruvate kinase (hLPYK) converts phosphoenolpyruvate to pyruvate in the final step of glycolysis. hLPYK is allosterically activated by fructose-1,6-bisphosphate (Fru-1,6-BP). The allosteric site, as defined by previous structural studies, is located in domain C between the phosphate-binding loop (residues 444-449) and the allosteric loop (residues 527-533). In this study, the X-ray crystal structures of four hLPYK variants were solved to make structural correlations with existing functional data. The variants are D499N, W527H, Á529/S531G (called GGG here) and S531E. The results revealed a conformational toggle between the open and closed positions of the allosteric loop. In the absence of Fru-1,6-BP the open position is stabilized, in part, by a cation-bond between Trp527 and Arg538 0 (from an adjacent monomer). In the S531E variant glutamate binds in place of the 6 0 -phosphate of Fru-1,6-BP in the allosteric site, leading to partial allosteric activation. Finally, the structure of the D499N mutant does not provide structural evidence for the previously observed allosteric activation of the D499N variant.

Cysteine dioxygenase (CDO) structurally resembles cupin enzymes that use a 3-His/1-Glu coordination scheme. However, the glutamate ligand is substituted with a cysteine (Cys93) residue, which forms a thioether bond with tyrosine (Tyr157) under physiological conditions. The reversion variant, C93E CDO, was generated in order to reestablish the more common 3-His/1-Glu metal ligands of the cupin superfamily. This variant provides a framework for testing the structural and functional significance of Cys93 and the cross-link in CDO. Although dioxygen consumption was observed with C93E CDO, it was not coupled with l-cysteine oxidation. Substrate analogues (d-cysteine, cysteamine, and 3-mercaptopropionate) were not viable substrates for the C93E CDO variant, although they showed variable coordinations to the iron center. The structures of C93E and cross-linked and non-cross-linked wild-type CDO were solved by X-ray crystallography to 1.91, 2.49, and 2.30 Å, respectively. The C93E CDO variant had similar overall structural properties compared to cross-linked CDO; however, the iron was coordinated by a 3-His/1-Glu geometry, leaving only two coordination sites available for dioxygen and bidentate l-cysteine binding. The hydroxyl group of Tyr157 shifted in both non-cross-linked and C93E CDO, and this displacement prevented the residue from participating in substrate stabilization. Based on these results, the divergence of the metal center of cysteine dioxygenase from the 3-His/1-Glu geometry seen with many cupin enzymes was essential for effective substrate binding. The substitution of Glu with Cys in CDO allows for a third coordination site on the iron for bidentate cysteine and monodentate oxygen binding.

The π-helix located at the tetramer interface of two-component FMN-dependent reductases contributes to the structural divergence from canonical FMN-bound reductases within the NADPH:FMN reductase family. The π-helix in the SsuE FMN-dependent reductase of the alkanesulfonate monooxygenase system has been proposed to be generated by the insertion of a Tyr residue in the conserved α4-helix. Variants of Tyr118 were generated, and their X-ray crystal structures determined, to evaluate how these alterations affect the structural integrity of the π-helix. The structure of the Y118A SsuE π-helix was converted to an α-helix, similar to the FMN-bound members of the NADPH:FMN reductase family. Although the π-helix was altered, the FMN binding region remained unchanged. Conversely, deletion of Tyr118 disrupted the secondary structural properties of the π-helix, generating a random coil region in the middle of helix 4. Both the Y118A and Δ118 SsuE SsuE variants crystallize as a dimer. The MsuE FMN reductase involved in the desulfonation of methanesulfonates is structurally similar to SsuE, but the π-helix contains a His insertional residue. Exchanging the π-helix insertional residue of each enzyme did not result in equivalent kinetic properties. Structure-based sequence analysis further demonstrated the presence of a similar Tyr residue in an FMN-bound reductase in the NADPH:FMN reductase family that is not sufficient to generate a π-helix. Results from the structural and functional studies of the FMN-dependent reductases suggest that the insertional residue alone is not solely responsible for generating the π-helix, and additional structural adaptions occur to provide the altered gain of function.

The Aspergillus fumigatus old yellow enzyme (OYE) EasA reduces chanoclavine-I aldehyde to dihydrochanoclavine aldehyde and works in conjunction with festuclavine synthase at the branchpoint for ergot alkaloid pathways. The crystal structure of the FMN-loaded EasA was determined to 1.8 Å resolution. The active-site amino acids of OYE are conserved, supporting a similar mechanism for reduction of the /-unsaturated aldehyde. The C-terminal tail of one monomer packs into the active site of a monomer in the next asymmetric unit, which is most likely to be a crystallization artifact and not a mechanism of self-regulation.