4 research outputs found

    <i>Streptomyces wadayamensis</i> MppP Is a Pyridoxal 5′-Phosphate-Dependent l‑Arginine α‑Deaminase, γ‑Hydroxylase in the Enduracididine Biosynthetic Pathway

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    l-Enduracididine (l-End) is a nonproteinogenic amino acid found in a number of bioactive peptides, including the antibiotics teixobactin, enduracidin, and mannopeptimycin. The potent activity of these compounds against antibiotic-resistant pathogens like MRSA and their novel mode of action have garnered considerable interest for the development of these peptides into clinically relevant antibiotics. This goal has been hampered, at least in part, by the fact that l-End is difficult to synthesize and not currently commercially available. We have begun to elucidate the biosynthetic pathway of this unusual building block. In mannopeptimycin-producing strains, like <i>Streptomyces wadayamensis</i>, l-End is produced from l-Arg by the action of three enzymes: MppP, MppQ, and MppR. Herein, we report the structural and functional characterization of MppP. This pyridoxal 5′-phosphate (PLP)-dependent enzyme was predicted to be a fold type I aminotransferase on the basis of sequence analysis. We show that MppP is actually the first example of a PLP-dependent hydroxylase that catalyzes a reaction of l-Arg with dioxygen to yield a mixture of 2-oxo-4-hydroxy-5-guanidinovaleric acid and 2-oxo-5-guanidinovaleric acid in a 1.7:1 ratio. The structure of MppP with PLP bound to the catalytic lysine residue (Lys221) shows that, while the tertiary structure is very similar to those of the well-studied aminotransferases, there are differences in the arrangement of active site residues around the cofactor that likely account for the unusual activity of this enzyme. The structure of MppP with the substrate analogue d-Arg bound shows how the enzyme binds its substrate and indicates why d-Arg is not a substrate. On the basis of this work and previous work with MppR, we propose a plausible biosynthetic scheme for l-End

    <i>Shewanella oneidensis</i> Cytochrome <i>c</i> Nitrite Reductase (ccNiR) Does Not Disproportionate Hydroxylamine to Ammonia and Nitrite, Despite a Strongly Favorable Driving Force

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    Cytochrome <i>c</i> nitrite reductase (ccNiR) from <i>Shewanella oneidensis</i>, which catalyzes the six-electron reduction of nitrite to ammonia <i>in vivo</i>, was shown to oxidize hydroxylamine in the presence of large quantities of this substrate, yielding nitrite as the sole free nitrogenous product. UV–visible stopped-flow and rapid-freeze-quench electron paramagnetic resonance data, along with product analysis, showed that the equilibrium between hydroxylamine and nitrite is fairly rapidly established in the presence of high initial concentrations of hydroxylamine, despite said equilibrium lying far to the left. By contrast, reduction of hydroxylamine to ammonia did not occur, even though disproportionation of hydroxylamine to yield both nitrite and ammonia is strongly thermodynamically favored. This suggests a kinetic barrier to the ccNiR-catalyzed reduction of hydroxylamine to ammonia. A mechanism for hydroxylamine reduction is proposed in which the hydroxide group is first protonated and released as water, leaving what is formally an NH<sub>2</sub><sup>+</sup> moiety bound at the heme active site. This species could be a metastable intermediate or a transition state but in either case would exist only if it were stabilized by the donation of electrons from the ccNiR heme pool into the empty nitrogen p orbital. In this scenario, ccNiR does not catalyze disproportionation because the electron-donating hydroxylamine does not poise the enzyme at a sufficiently low potential to stabilize the putative dehydrated hydroxylamine; presumably, a stronger reductant is required for this

    Metabolic Function for Human Renalase: Oxidation of Isomeric Forms of β‑NAD(P)H that Are Inhibitory to Primary Metabolism

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    Renalase is a recently identified flavoprotein that has been associated with numerous physiological maladies. There remains a prevailing belief that renalase functions as a hormone, imparting an influence on vascular tone and heart rate by oxidizing circulating catecholamines, chiefly epinephrine. This activity, however, has not been convincingly demonstrated in vitro, nor has the stoichiometry of this transformation been shown. In prior work we demonstrated that renalase induced rapid oxidation of low-level contaminants of β-NAD­(P)H solutions (Beaupre, B. A. et al. (2013) Biochemistry 52, 8929−8937; Beaupre, B. A. et al. (2013) J. Am. Chem. Soc. 135, 13980–13987). Slow aqueous speciation of β-NAD­(P)H resulted in the production of renalase substrate molecules whose spectrophotometric characteristics and equilibrium fractional accumulation closely matched those reported for α-anomers of NAD­(P)­H. The fleeting nature of these substrates precluded structural assignment. Here we structurally assign and identify two substrates for renalase. These molecules are 2- and 6-dihydroNAD­(P), isomeric forms of β-NAD­(P)H that arise either by nonspecific reduction of β-NAD­(P)<sup>+</sup> or by tautomerization of β-NAD­(P)­H (4-dihydroNAD­(P)). The pure preparations of these molecules induce rapid reduction of the renalase flavin cofactor (230 s<sup>–1</sup> for 6-dihydroNAD, 850 s<sup>–1</sup> for 2-dihydroNAD) but bind only a few fold more tightly than β-NADH. We also show that 2- and 6-dihydroNAD­(P) are potent inhibitors of primary metabolism dehydrogenases and therefore conclude that the metabolic function of renalase is to oxidize these isomeric NAD­(P)H molecules to β-NAD­(P)<sup>+</sup>, eliminating the threat they pose to normal respiratory activity

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

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    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 (<i>m</i>enaquinone, <i>s</i>iderophore, and <i>t</i>ryptophan 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. The isomerase enzymes release isochorismate or aminodeoxychorismate as the product, while the synthase enzymes also have lyase activity that displaces pyruvate to form either salicylate or anthranilate. This has led to the hypothesis that the isomerase and lyase activities performed by the MST enzymes are functionally conserved. Here we have developed tailored pre-steady-state approaches to establish the kinetic mechanisms of the isochorismate and salicylate synthase enzymes of siderophore biosynthesis. Our data are centered on the role of magnesium ions, which inhibit the isochorismate synthase enzymes but not the salicylate synthase enzymes. Prior structural data have suggested that binding of the metal ion occludes access or egress of substrates. Our kinetic data indicate that for the production of isochorismate, a high magnesium ion concentration suppresses the rate of release of product, accounting for the observed inhibition and establishing the basis of the ordered-addition kinetic mechanism. Moreover, we show that isochorismate is channeled through the synthase reaction as an intermediate that is retained in the active site by the magnesium ion. Indeed, the lyase-active enzyme has 3 orders of magnitude higher affinity for the isochorismate complex relative to the chorismate complex. Apparent negative-feedback inhibition by ferrous ions is documented at nanomolar concentrations, which is a potentially physiologically relevant mode of regulation for siderophore biosynthesis in vivo
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