Kinetic and structural characterization of carboxyspermidine dehydrogenase of polyamine biosynthesis.

Polyamines are positively charged alkylamines ubiquitous among eukaryotes, prokaryotes, and archaea. Humans obtain polyamines through dietary intake, metabolic production, or uptake of polyamines made by gut microbes. The polyamine biosynthetic pathway used by most gut microbes differs from that used by human cells.

The 2.4 Å structure of Zymomonas mobilis pyruvate kinase: Implications for stability and regulation.

Human liver pyruvate kinase (hlPYK) catalyzes the final step in glycolysis, the formation of pyruvate (PYR) and ATP from phosphoenolpyruvate (PEP) and ADP. Fructose 1,6-bisphosphate (FBP), a pathway intermediate of glycolysis, serves as an allosteric activator of hlPYK. Zymomonas mobilis pyruvate kinase (ZmPYK) performs the final step of the Entner-Doudoroff pathway, which is similar to glycolysis in that energy is harvested from glucose and pyruvate is generated.

Staphylopine and pseudopaline dehydrogenase from bacterial pathogens catalyze reversible reactions and produce stereospecific metallophores.

Pseudopaline and staphylopine are opine metallophores biosynthesized by and , respectively. The final step in opine metallophore biosynthesis is the condensation of the product of a nicotianamine (NA) synthase reaction ( l-HisNA for pseudopaline and d-HisNA for staphylopine) with an α-keto acid (α-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 ()-stereochemistry at carbon C2 of the α-keto acid (prochiral prior to catalysis).

Changes in the allosteric site of human liver pyruvate kinase upon activator binding include the breakage of an intersubunit cation-π bond.

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).

Stuffed Methyltransferase Catalyzes the Penultimate Step of Pyochelin Biosynthesis.

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.

Not as easy as π: An insertional residue does not explain the π-helix gain-of-function in two-component FMN reductases.

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.

Staphylopine, pseudopaline, and yersinopine dehydrogenases: A structural and kinetic analysis of a new functional class of opine dehydrogenase.

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.

Biosynthesis of an Opine Metallophore by Pseudomonas aeruginosa.

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.