Presence of a plant-like proton-pumping pyrophosphatase in acidocalcisomes of Trypanosoma cruzi.
AuthorsScott DA, de Souza W, Benchimol M, Zhong L, Lu HG, Moreno SN, Docampo R
JournalJ Biol Chem
PubMed ID9705361
'The vacuolar-type proton-translocating pyrophosphatase (V-H+-PPase) is an enzyme previously described in detail only in plants. This paper demonstrates its presence in the trypanosomatid Trypanosoma cruzi. Pyrophosphate promoted organellar acidification in permeabilized amastigotes, epimastigotes, and trypomastigotes of T. cruzi. This activity was stimulated by K+ ions and was inhibited by Na+ ... More
A broadly applicable continuous spectrophotometric assay for measuring aminoacyl-tRNA synthetase activity.
AuthorsLloyd AJ, Thomann HU, Ibba M, Söll D
JournalNucleic Acids Res
PubMed ID7659511
'We describe a convenient, simple and novel continuous spectrophotometric method for the determination of aminoacyl-tRNA synthetase activity. The assay relies upon the measurement of inorganic pyrophosphate generated in the first step of the aminoacylation of a tRNA. Pyrophosphate release is coupled to inorganic pyrophosphatase, to generate phosphate, which in turn ... More
Thiamin biosynthesis in Escherichia coli. Identification of this thiocarboxylate as the immediate sulfur donor in the thiazole formation.
ThiFSGH and ThiI are required for the biosynthesis of the thiazole moiety of thiamin in Escherichia coli. The overproduction, purification, and characterization of ThiFS and the identification of two of the early steps in the biosynthesis of the thiazole moiety of thiamin are described here. ThiS isolated from E. coli ... More
AcsD catalyzes enantioselective citrate desymmetrization in siderophore biosynthesis.
AuthorsSchmelz S, Kadi N, McMahon SA, Song L, Oves-Costales D, Oke M, Liu H, Johnson KA, Carter LG, Botting CH, White MF, Challis GL, Naismith JH,
JournalNat Chem Biol
PubMed ID19182782
Bacterial pathogens need to scavenge iron from their host for growth and proliferation during infection. They have evolved several strategies to do this, one being the biosynthesis and excretion of small, high-affinity iron chelators known as siderophores. The biosynthesis of siderophores is an important area of study, not only for ... More
A spectrophotometric method to measure enzymatic activity in reactions that generate inorganic pyrophosphate.
AuthorsUpson RH, Haugland RP, Malekzadeh MN, Haugland RP
JournalAnal Biochem
PubMed ID8954523
This paper describes a spectrophotometric assay that can measure the inorganic pyrophosphate produced from various enzymatic reactions. This is a coupled assay in which the addition of inorganic pyrophosphatase initially cleaves the pyrophosphate into two molecules of phosphate. The phosphate is then detected by the conversion of 2-amino-6-mercapto-7-methylpurine ribonucleoside to ... More
Cloning and characterization of a Streptomyces single module type non-ribosomal peptide synthetase catalyzing a blue pigment synthesis.
AuthorsTakahashi H, Kumagai T, Kitani K, Mori M, Matoba Y, Sugiyama M
JournalJ Biol Chem
PubMed ID17237222
In the present study, we cloned a gene, designated bpsA, which encodes a single module type non-ribosomal peptide synthetase (NRPS) from a D-cycloserine (DCS)-producing Streptomyces lavendulae ATCC11924. A putative oxidation domain is significantly integrated into the adenylation domain of the NRPS, and the condensation domain is absent from the module. ... More
Characterization of isolated acidocalcisomes of Trypanosoma cruzi.
AuthorsScott DA, Docampo R
JournalJ Biol Chem
PubMed ID10816577
The acidocalcisome is an acidic calcium store in trypanosomatids with a vacuolar-type proton-pumping pyrophosphatase (V-H(+)-PPase) located in its membrane. In this paper, we describe a new method using iodixanol density gradients for purification of the acidocalcisome from Trypanosoma cruzi epimastigotes. Pyrophosphatase assays indicated that the isolated organelle was at least ... More
Equilibrium and kinetic studies of substrate binding to 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase from Escherichia coli.
AuthorsBermingham A, Bottomley JR, Primrose WU, Derrick JP
JournalJ Biol Chem
PubMed ID10751386
6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the pyrophosphorylation of 6-hydroxymethyl-7,8-dihydropterin (HMDP) by ATP to form 6-hydroxymethyl-7,8-dihydropterin pyrophosphate, an intermediate in the pathway for folic acid biosynthesis. The enzyme has been identified as a potential target for antimicrobial drugs. Equilibrium binding studies showed that Escherichia coli HPPK-bound ATP or the nonhydrolyzable ATP analogue ... More
Vibriobactin biosynthesis in Vibrio cholerae: VibH is an amide synthase homologous to nonribosomal peptide synthetase condensation domains.
AuthorsKeating TA, Marshall CG, Walsh CT
JournalBiochemistry
PubMed ID11112537
The Vibrio cholerae siderophore vibriobactin is biosynthesized from three molecules of 2,3-dihydroxybenzoate (DHB), two molecules of L-threonine, and one of norspermidine. Of the four genes positively implicated in vibriobactin biosynthesis, we have here expressed, purified, and assayed the products of three: vibE, vibB, and vibH. All three are homologous to ... More
Selectivity of the yersiniabactin synthetase adenylation domain in the two-step process of amino acid activation and transfer to a holo-carrier protein domain.
AuthorsKeating TA, Suo Z, Ehmann DE, Walsh CT
JournalBiochemistry
PubMed ID10694396
The adenylation (A) domain of the Yersinia pestis nonribosomal peptide synthetase that biosynthesizes the siderophore yersiniabactin (Ybt) activates three molecules of L-cysteine and covalently aminoacylates the phosphopantetheinyl (P-pant) thiols on three peptidyl carrier protein (PCP) domains embedded in the two synthetase subunits, two in cis (PCP1, PCP2) in subunit HMWP2 ... More