caa a22 4500 445321776 CHVBK 20180317142706.0 cr unu---uuuuu 170323e20110801xx s 000 0 eng 10.1007/s10534-011-9416-7 doi (NATIONALLICENCE)springer-10.1007/s10534-011-9416-7 Genetic and biochemical analyses of chromosome and plasmid gene homologues encoding ICL and ArCP domains in Vibrio anguillarum strain 775 [Elektronische Daten] [Manuela Di Lorenzo, Michiel Stork, Jorge Crosa] Anguibactin, the siderophore produced by Vibrio anguillarum 775 is synthesized from 2,3-dihydroxybenzoic acid (DHBA), cysteine and hydroxyhistamine via a nonribosomal peptide synthetase (NRPS) mechanism. Most of the genes encoding anguibactin biosynthetic proteins are harbored by the pJM1 plasmid. In this work we report the identification of a homologue of the plasmid-encoded angB on the chromosome of strain 775. The product of both genes harbor an isochorismate lyase (ICL) domain that converts isochorismic acid to 2,3-dihydro-2,3-dihydroxybenzoic acid, one of the steps of DHBA synthesis. We show in this work that both ICL domains are functional in the production of DHBA in V. anguillarum as well as in E. coli. Substitution by alanine of the aspartic acid residue in the active site of both ICL domains completely abolishes their isochorismate lyase activity in vivo. The two proteins also carry an aryl carrier protein (ArCP) domain. In contrast with the ICL domains only the plasmid encoded ArCP can participate in anguibactin production as determined by complementation analyses and site-directed mutagenesis in the active site of the plasmid encoded protein, S248A. The site-directed mutants, D37A in the ICL domain and S248A in the ArCP domain of the plasmid encoded AngB were also tested in vitro and clearly show the importance of each residue for the domain function and that each domain operates independently. Springer Science+Business Media, LLC., 2011 Iron nationallicence Siderophore nationallicence Nonribosomal peptide synthetase nationallicence 2,3-dihydroxybenzoic acid nationallicence Isochorismate lyase domain nationallicence Aryl carrier protein domain nationallicence Di Lorenzo Manuela Department of Microbial Ecology, Netherlands Institute of Ecology, Wageninegen, The Netherlands aut Stork Michiel Department of Molecular Microbiology, Utrecht University, Utrecht, The Netherlands aut Crosa Jorge Department of Molecular Microbiology and Immunology, Oregon Health and Science University, 97239-3098, Portland, OR, USA aut BioMetals Springer Netherlands 24/4(2011-08-01), 629-643 0966-0844 24:4<629 2011 24 10534 https://doi.org/10.1007/s10534-011-9416-7 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s10534-011-9416-7 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Di Lorenzo Manuela Department of Microbial Ecology, Netherlands Institute of Ecology, Wageninegen, The Netherlands aut NATIONALLICENCE 700 1- Stork Michiel Department of Molecular Microbiology, Utrecht University, Utrecht, The Netherlands aut NATIONALLICENCE 700 1- Crosa Jorge Department of Molecular Microbiology and Immunology, Oregon Health and Science University, 97239-3098, Portland, OR, USA aut NATIONALLICENCE 773 0- BioMetals Springer Netherlands 24/4(2011-08-01), 629-643 0966-0844 24:4<629 2011 24 10534 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer