A continuous fluorescence assay to measure nicotianamine synthase activity.

S-adenosylmethionine (SAM) is most widely known as the biological methylating agent of methyltransferases and for generation of radicals by the iron-sulfur dependent Radical SAM enzymes. SAM also serves as a substrate in biosynthetic reactions that harvest the aminobutyrate moiety of the methionine, producing methylthioadenosine as a co-product. These reactions are found in the production of polyamines such as spermine, siderophores derived from nicotianamine, and opine metallophores staphylopine and pseudopaline, among others.

Kinetic and Structural Characterization of Hypoxanthine-Guanine-Xanthine Phosphoribosyltransferases and Repurposing of Transition-State Analogue Inhibitors.

Over 70 million people are currently at risk of developing Chagas Disease (CD) infection, with more than 8 million people already infected worldwide. Current treatments are limited and innovative therapies are required. , the etiological agent of CD, is a purine auxotroph that relies on phosphoribosyltransferases to salvage purine bases from their hosts for the formation of purine nucleoside monophosphates.

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.

Evidence for the Chemical Mechanism of RibB (3,4-Dihydroxy-2-butanone 4-phosphate Synthase) of Riboflavin Biosynthesis.

RibB (3,4-dihydroxy-2-butanone 4-phosphate synthase) is a magnesium-dependent enzyme that excises the C4 of d-ribulose-5-phosphate (d-Ru5P) as formate. RibB generates the four-carbon substrate for lumazine synthase that is incorporated into the xylene moiety of lumazine and ultimately the riboflavin isoalloxazine. The reaction was first identified by Bacher and co-workers in the 1990s, and their chemical mechanism hypothesis became canonical despite minimal direct evidence.

Rational inhibitor design for Pseudomonas aeruginosa salicylate adenylation enzyme PchD.

Pseudomonas aeruginosa is an increasingly antibiotic-resistant pathogen that causes severe lung infections, burn wound infections, and diabetic foot infections. P. aeruginosa produces the siderophore pyochelin through the use of a non-ribosomal peptide synthetase (NRPS) biosynthetic pathway.

Substitutions at a rheostat position in human aldolase A cause a shift in the conformational population.

Some protein positions play special roles in determining the magnitude of protein function: at such "rheostat" positions, varied amino acid substitutions give rise to a continuum of functional outcomes, from wild type (or enhanced), to intermediate, to loss of function. This observed range raises interesting questions about the biophysical bases by which changes at single positions have such varied outcomes. Here, we assessed variants at position 98 in human aldolase A ("I98X").

Finding Ways to Relax: A Revisionistic Analysis of the Chemistry of GTP Cyclohydrolase II.

Guanosine triphosphate (GTP) cyclohydrolase II (RibA) is one of three enzymes that hydrolytically cleave the C8-N9 bond of the GTP guanine. RibA also catalyzes a subsequent hydrolytic attack at the base liberating formate and in addition cleaves the α-β phosphodiester bond of the triphosphate to form pyrophosphate (PPi). These hydrolytic reactions are promoted by tandem active-site metal ions, zinc and magnesium, that respectively function at the GTP guanine and triphosphate moieties.

The 15-Amino Acid Repeat Region of Adenomatous Polyposis Coli Is Intrinsically Disordered and Retains Conformational Flexibility upon Binding β-Catenin.

The tumor suppressor Adenomatous polyposis coli (APC) is a large, multidomain protein with many identified cellular functions. The best characterized role of APC is to scaffold a protein complex that negatively regulates Wnt signaling via β-catenin destruction. This destruction is mediated by β-catenin binding to centrally located 15- and 20-amino acid repeat regions of APC.

The 3-His Metal Coordination Site Promotes the Coupling of Oxygen Activation to Cysteine Oxidation in Cysteine Dioxygenase.

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.

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).

PvdF of pyoverdin biosynthesis is a structurally unique N-formyltetrahydrofolate-dependent formyltransferase.

The hydroxyornithine transformylase from Pseudomonas aeruginosa is known by the gene name pvdF, and has been hypothesized to use N-formyltetrahydrofolate (N-fTHF) as a co-substrate formyl donor to convert N-hydroxyornithine (OHOrn) to N-formyl- N-hydroxyornithine (fOHOrn). PvdF is in the biosynthetic pathway for pyoverdin biosynthesis, a siderophore generated under iron-limiting conditions that has been linked to virulence, quorum sensing and biofilm formation. The structure of PvdF was determined by X-ray crystallography to 2.

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.

Unraveling the Structure and Mechanism of the MST(ery) Enzymes.

The menaquinone, siderophore, and tryptophan (MST) enzymes transform chorismate to generate precursor molecules for the biosynthetic pathways defined in their name. Kinetic data, both steady-state and transient-state, and X-ray crystal structures indicate that these enzymes are highly conserved both in mechanism and in structure. Because these enzymes are found in pathogens but not in humans, there is considerable interest in these enzymes as drug design targets.

Nonribosomal peptides for iron acquisition: pyochelin biosynthesis as a case study.

Microbes synthesize small, iron-chelating molecules known as siderophores to acquire iron from the environment. One way siderophores are generated is by nonribosomal peptide synthetases (NRPSs). The bioactive peptides generated by NRPS enzymes have unique chemical features, which are incorporated by accessory and tailoring domains or proteins.

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.

Ligand binding phenomena that pertain to the metabolic function of renalase.

Renalase catalyzes the oxidation of isomers of β-NAD(P)H that carry the hydride in the 2 or 6 positions of the nicotinamide base to form β-NAD(P). This activity is thought to alleviate inhibition of multiple β-NAD(P)-dependent enzymes of primary and secondary metabolism by these isomers. Here we present evidence for a variety of ligand binding phenomena relevant to the function of renalase.

Holo Structure and Steady State Kinetics of the Thiazolinyl Imine Reductases for Siderophore Biosynthesis.

Thiazolinyl imine reductases catalyze the NADPH-dependent reduction of a thiazoline to a thiazolidine, a required step in the formation of the siderophores yersiniabactin (Yersinia spp.) and pyochelin (Pseudomonas aeruginosa). These stand-alone nonribosomal peptide tailoring domains are structural homologues of sugar oxidoreductases.

An Open and Shut Case: The Interaction of Magnesium with MST Enzymes.

The shikimate pathway of bacteria, fungi, and plants generates chorismate, which is drawn into biosynthetic pathways that form aromatic amino acids and other important metabolites, including folates, menaquinone, and siderophores. Many of the pathways initiated at this branch point transform chorismate using an MST enzyme. The MST enzymes (menaquinone, siderophore, and tryptophan biosynthetic enzymes) are structurally homologous and magnesium-dependent, and all perform similar chemical permutations to chorismate by nucleophilic addition (hydroxyl or amine) at the 2-position of the ring, inducing displacement of the 4-hydroxyl.

Breaking a pathogen's iron will: Inhibiting siderophore production as an antimicrobial strategy.

The rise of antibiotic resistance is a growing public health crisis. Novel antimicrobials are sought, preferably developing nontraditional chemical scaffolds that do not inhibit standard targets such as cell wall synthesis or the ribosome. Iron scavenging has been proposed as a viable target, because bacterial and fungal pathogens must overcome the nutritional immunity of the host to be virulent.

You are lost without a map: Navigating the sea of protein structures.

X-ray crystal structures propel biochemistry research like no other experimental method, since they answer many questions directly and inspire new hypotheses. Unfortunately, many users of crystallographic models mistake them for actual experimental data. Crystallographic models are interpretations, several steps removed from the experimental measurements, making it difficult for nonspecialists to assess the quality of the underlying data.

Structure of an Aspergillus fumigatus old yellow enzyme (EasA) involved in ergot alkaloid biosynthesis.

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.

Expanding the results of a high throughput screen against an isochorismate-pyruvate lyase to enzymes of a similar scaffold or mechanism.

Antibiotic resistance is a growing health concern, and new avenues of antimicrobial drug design are being actively sought. One suggested pathway to be targeted for inhibitor design is that of iron scavenging through siderophores. Here we present a high throughput screen to the isochorismate-pyruvate lyase of Pseudomonas aeruginosa, an enzyme required for the production of the siderophore pyochelin.