The dhb operon of Bacillus subtilis encodes the biosynthetic template for the catecholic siderophore 2,3-dihydroxybenzoate-glycine-threonine trimeric ester bacillibactin.
Bacillus subtilis was reported to produce the catecholic siderophore itoic acid (2,3-dihydroxybenzoate (DHB)-glycine) in response to iron deprivation. However, by inspecting the DNA sequences of the genes dhbE, dhbB, and dhbF as annotated by the B. subtilis genome project to encode the synthetase complex for the siderophore assembly, various sequence errors within the dhbF gene were predicted and confirmed by re-sequencing. According to the corrected sequence, dhbF encodes a dimodular instead of a monomodular nonribosomal peptide synthetase. We have heterologously expressed, purified, and assayed the substrate selectivity of the recombinant proteins DhbB, DhbE, and DhbF. DhbE, a stand-alone adenylation domain of 59.9 kDa, activates, in an ATP-dependent reaction, DHB, which is subsequently transferred to the free thiol group of the cofactor phosphopantetheine of the bifunctional isochorismate lyase/aryl carrier protein DhbB. The third synthetase, DhbF, is a dimodular nonribosomal peptide synthetase of 264 kDa that specifically adenylates threonine and, to a lesser extent, glycine and that covalently loads both amino acids onto their corresponding peptidyl carrier domains. To functionally link the dhb gene cluster to siderophore synthesis, we have disrupted the dhbF gene. Comparative mass spectrometric analysis of culture extracts from both the wild type and the dhbF mutant led to the identification of a mass peak at m/z 881 ([M-H](1-)) that corresponds to a cyclic trimeric ester of DHB-glycine-threonine.
May
J J
JJ
Department of Chemistry, Philipps-Universität Marburg, D-35032 Marburg, Germany.
Wendrich
T M
TM
Marahiel
M A
MA
D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000596:Amino Acids / Q000737:chemistry; D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004952:Esters / Q000737:chemistry*; D062385:Hydroxybenzoates / Q000737:chemistry* / Q000494:pharmacology; D007202:Indicators and Reagents / Q000494:pharmacology; D013058:Mass Spectrometry; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D009876:Operon; D010957:Plasmids / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D017262:Siderophores / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry
D000596:Amino Acids; D004952:Esters; D062385:Hydroxybenzoates; D007202:Indicators and Reagents; D009842:Oligopeptides; D011994:Recombinant Proteins; D017262:Siderophores; D013438:Sulfhydryl Compounds; C430721:bacillibactin; C015076:chrome azurol S; D000249:Adenosine Monophosphate; C009135:2,3-dihydroxybenzoic acid; D000255:Adenosine Triphosphate