tailieunhanh - Báo cáo khoa học: Mutational analysis of the C-domain in nonribosomal peptide synthesis

The initial condensation event in the nonribosomal biosyn-thesisof thepeptideantibioticsgramicidinSandtyrocidineA takes place between a phenylalanine activating racemase GrsA/TycAand the ®rst proline-activatingmodule ofGrsB/ TycB. Recently we established a minimalin vitro model system for NRPS with recombinant His6-tagged GrsA (GrsA Phe-ATE; 127 kDa) and TycB1 (TycB1Pro-CAT; 120 kDa) and demonstrated the catalytic function of the C-domaininTycB1Pro-CAT to form a peptide bond between phenylalanine and proline during diketopiperazine formation (DKP). . | Eur. J. Biochem. 269 620-629 2002 FEBS 2002 Mutational analysis of the C-domain in nonribosomal peptide synthesis Veit Bergendahl Uwe Linne and Mohamed A. Marahiel Biochemie Fachbereich Chemie Philipps-UniversitiÈt Marburg Germany The initial condensation event in the nonribosomal biosynthesis of the peptide antibiotics gramicidin S and tyrocidine A takes place between a phenylalanine activating racemase GrsA TycA and the first proline-activating module of GrsB TycB. Recently we established a minimal in vitro model system for NRPS with recombinant His6-tagged GrsA GrsAPhe-ATE 127 kDa and TycBl TycB1Pro-CAT 120 kDa and demonstrated the catalytic function of the C-domain in TycB1Pro-CAT to form a peptide bond between phenylalanine and proline during diketopiperazine formation DKP . In this work we took advantage of this system to identify catalytically important residues in the C-domain of TycB1Pro-CAT using site-directed mutagenesis and peptide mapping. Mutations in TycB1Pro-CAT of 10 strictly conserved residues among 80 other C-domains with potential catalytic function revealed that only R62A H147R and D151N are impaired in peptide-bond formation. All other mutations led to either unaffected Q19A C154A S Y166F W and R284A or insoluble proteins H146A R67A and W202L . Although 100 nM of the serine protease inhibitors N-a-tosyl-L-phenylalanylchlo-romethane or phenylmethanesulfonyl fluoride completely abolished DKP synthesis no covalently bound inhibitor derivatives in the C-domain could be identifed by peptide mapping using HPLC-MS. Though the results do not reveal a particular mechanism for the C-domain they exhibit a possible way of catalysis analogous to the functionally related enzymes chloramphenicol acetyltransferase and dihydrolipoyl transacetylase. Based on this we propose a mechanism in which one catalytic residue H147 and two other structural residues R62 and D151 are involved in amino-acid condensation. Keywords nonribosomal peptide synthesis nonribosomal .

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