Opine dehydrogenases (ODHs) from the bacterial pathogens ,, and perform the final enzymatic step in the biosynthesis of a new class of opine metallophores, which includes staphylopine, pseudopaline, and yersinopine, respectively. Growing evidence indicates an important role for this pathway in metal acquisition and virulence, including in lung and burn-wound infections () and in blood and heart infections (). Here, we present kinetic and structural characterizations of these three opine dehydrogenases. A steady-state kinetic analysis revealed that the three enzymes differ in α-keto acid and NAD(P)H substrate specificity and nicotianamine-like substrate stereoselectivity. The structural basis for these differences was determined from five ODH X-ray crystal structures, ranging in resolution from 1.9 to 2.5 Å, with or without NADP bound. Variation in hydrogen bonding with NADPH suggested an explanation for the differential recognition of this substrate by these three enzymes. Our analysis further revealed candidate residues in the active sites required for binding of the α-keto acid and nicotianamine-like substrates and for catalysis. This work reports the first structural kinetic analyses of enzymes involved in opine metallophore biosynthesis in three important bacterial pathogens of humans.

Bacterial pathogenesis frequently requires metal acquisition by specialized, small-molecule metallophores. We hypothesized that the Gram-negative Pseudomonas aeruginosa encodes the enzymes nicotianamine synthase (NAS) and opine dehydrogenase (ODH), biosynthesizing a new class of opine metallophore, previously characterized only in the unrelated Gram-positive organism Staphylococcus aureus. The identity of this metallophore, herein named pseudopaline, was determined through measurements of binding affinity, the in vitro reconstitution of the biosynthetic pathway to screen potential substrates, and the confirmation of product formation by mass spectrometry. Pseudopaline and the S. aureus metallophore staphylopine exhibit opposite stereochemistry for the histidine moiety, indicating unique recognition by NAS. Additionally, we demonstrate SaODH catalysis in the presence of pyruvate, as previously shown, but also oxaloacetate, suggesting the potential for the production of a variant form of staphylopine, while PaODH specifically recognizes α-ketoglutarate. Both the staphylopine and pseudopaline operons have been implicated in the pathogenesis of key infectious disease states and warrant further study.

Edited by Ruma Banerjee Pseudopaline and staphylopine are opine metallophores biosynthesized by Pseudomonas aeruginosa and Staphylococcus aureus, respectively. The final step in opine metallophore biosynthesis is the condensation of the product of a nicotianamine (NA) synthase reaction (i.e. L-HisNA for pseudopaline and D-HisNA for staphylopine) with an ␣-keto acid (␣-ketoglutarate for pseudopaline and pyruvate for staphylopine), which is performed by an opine dehydrogenase. We hypothesized that the opine dehydrogenase reaction would be reversible only for the opine metallophore product with (R)-stereochemistry at carbon C2 of the ␣-keto acid (prochiral prior to catalysis). A kinetic analysis using stopped-flow spectrometry with (R)-or (S)-staphylopine and kinetic and structural analysis with (R)-and (S)-pseudopaline confirmed catalysis in the reverse direction for only (R)-staphylopine and (R)-pseudopaline, verifying the stereochemistry of these two opine metallophores. Structural analysis at 1.57-1.85 Å resolution captured the hydrolysis of (R)-pseudopaline and allowed identification of a binding pocket for the L-histidine moiety of pseudopaline formed through a repositioning of Phe-340 and Tyr-289 during the catalytic cycle. Transient-state kinetic analysis revealed an ordered release of NADP ؉ followed by staphylopine, with staphylopine release being the rate-limiting step in catalysis. Knowledge of the stereochemistry for opine metallophores has implications for future studies involving kinetic analysis, as well as opine metallophore transport, metal coordination, and the generation of chiral amines for pharmaceutical development.

No abstract

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.

Nonribosomal peptide synthetases use tailoring domains to incorporate chemical diversity into the final natural product. A structurally unique set of tailoring domains are found to be stuffed within adenylation domains and have only recently begun to be characterized. PchF is the NRPS termination module in pyochelin biosynthesis and includes a stuffed methyltransferase domain responsible for S-adenosylmethionine (AdoMet) dependent N-methylation. Recent studies of stuffed methyltransferase domains propose a model in which methylation occurs on amino acids after adenylation and thiolation rather than after condensation to the nascent peptide chain. Herein, we characterize the adenylation and stuffed methyltransferase didomain of PchF through the synthesis and use of substrate analogs, steady-state kinetics, and onium chalcogen effects. We provide evidence that methylation occurs through an S N 2 reaction after thiolation, condensation, cyclization and reduction of the module substrate cysteine and is the penultimate step in pyochelin biosynthesis.

Traditionally, furfuryl alcohol (FOL) is produced from biomass-derived furfural (FAL) by hydrogenation using metal-based chemocatalysts. It is challenging due to high metal toxicity, high hydrogen partial pressure, and the need for organic solvents. NAD­(P)­H-dependent yeast alcohol dehydrogenase I (YADH), which offers high atom economy and up to 100% product selectivity at room temperature in an aqueous medium, is used in this study for the biocatalytic production of FOL from FAL using ethanol (EtOH) as the terminal reductant for in situ regeneration of NAD­(P)­H. Up to 74% FAL conversion was observed at pH 8 with 40 and 160 mM initial FAL and EtOH concentrations, respectively. The conversion was determined to be equilibrium-limited. Circular dichroism spectroscopy and differential scanning fluorimetry studies of YADH show a significant change in the secondary structure upon treatment with increasing concentrations of aldehydes resulting in loss of catalytic activity. Benign reaction conditions support efforts toward sustainable processing, but opportunities for further improvement by increasing product titer and catalyst stability have been identified. This study lays the framework for developing the science and process for alternatives to biomass-derived ethanol.

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.