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Titolo:
Cofactor requirements and reconstitution of Microcin B17 synthetase: A multienzyme complex that catalyzes the formation of oxazoles and thiazoles in the antibiotic Microcin B17
Autore:
Milne, JC; Roy, RS; Eliot, AC; Kelleher, NL; Wokhlu, A; Nickels, B; Walsh, CT;
Indirizzi:
Harvard Univ, Sch Med, Dept Biol Chem & Mol Pharmacol, Boston, MA 02115 USA Harvard Univ Boston MA USA 02115 em & Mol Pharmacol, Boston, MA 02115 USA
Titolo Testata:
BIOCHEMISTRY
fascicolo: 15, volume: 38, anno: 1999,
pagine: 4768 - 4781
SICI:
0006-2960(19990413)38:15<4768:CRAROM>2.0.ZU;2-I
Fonte:
ISI
Lingua:
ENG
Soggetto:
DNA-GYRASE INHIBITOR; MUTATIONAL ANALYSIS; STRUCTURE ELUCIDATION; CYTIDINE DEAMINASE; PEPTIDE; BINDING; SEQUENCE; RECOGNITION; CLEAVAGE; PROTEIN;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
49
Recensione:
Indirizzi per estratti:
Indirizzo: Walsh, CT Harvard Univ, Sch Med, Dept Biol Chem & Mol Pharmacol, Boston, MA 02115 USA Harvard Univ Boston MA USA 02115 harmacol, Boston, MA 02115 USA
Citazione:
J.C. Milne et al., "Cofactor requirements and reconstitution of Microcin B17 synthetase: A multienzyme complex that catalyzes the formation of oxazoles and thiazoles in the antibiotic Microcin B17", BIOCHEM, 38(15), 1999, pp. 4768-4781

Abstract

In the maturation of the Escherichia coli antibiotic Microcin B17 (MccB 17), the McbA preproantibiotic is modified post-translationally by the multimeric microcin synthetase complex (composed of the McbB, -C, and -D proteins), which cyclizes four cysteines and four serines to thiazoles and oxazoles, respectively. Herein, we report the purification of individual subunits of MccB 17 synthetase as fusions to maltose binding protein (MBP), and the in vitro reconstitution of heterocyclization activity. Preliminary characterization of each subunit reveals McbB to be a zinc-containing protein that may catalyze the initial cyclodehydration step, and McbC to contain flavin, consistent with an anticipated role for a dehydrogenase. We have previouslydemonstrated that McbD is a regulated ATPase/GTPase that may function as aconformational switch. Photolabeling experiments with the McbA propeptide now identify McbD as the initial site of substrate recognition. Heterocyclization activity was reconstituted only by combining all three subunits, demonstrating that each protein is required for heterocycle formation. Titration assays indicate that the subunits bind to each other with at least micromolar affinities, although McbD affords activity only after the MBP tag is proteolytically removed. Subunit competition assays with an McbD(D147A) mutant, which yields a catalytically deficient synthetase in vivo, show it to be defective in complex formation, whereas the McbB(C181A/C184A) double mutant, which is also inactive, competitively inhibits reconstitution by native McbB. Addition of the HtpG chaperone (originally shown to copurify with MccB17 synthetase), does not stimulate synthetase reconstitution or heterocyclization activity in vitro. A model for synthetase activity is proposed.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 05/07/20 alle ore 03:31:41