tailieunhanh - Báo cáo khoa học: Mutational analysis of a type II thioesterase associated with nonribosomal peptide synthesis

Recent studies on type II thioesterases (TEIIs) involved in microbial secondary metabolism described a role for these enzymes in the removal of short acyl-S- phosphopantetheine intermediates from misprimed holo-(acyl carrier proteins) and holo-(peptidyl carrier proteins) of polyketide synthases and nonribosomal peptide synthetases. Because of the absence of structural information on this class of enzymes, we performed a mutational analysis on a prototype TEII essential for efficient productionof the lipopeptide antibiotic surfactin (TEIIsrf), which led to identificationof catalytic and structural residues. . | Eur. J. Biochem. 271 1536-1545 2004 FEBS 2004 doi Mutational analysis of a type II thioesterase associated with nonribosomal peptide synthesis Uwe Linne Dirk Schwarzer Gunnar N. Schroeder and Mohamed A. Marahiel Philipps Universitat Marburg Fachbereich Chemie Biochemie Germany Recent studies on type II thioesterases TEIIs involved in microbial secondary metabolism described a role for these enzymes in the removal of short acyl-S- phosphopantetheine intermediates from misprimed holo- acyl carrier proteins and holo- peptidyl carrier proteins of polyketide synthases and nonribosomal peptide synthetases. Because of the absence of structural information on this class of enzymes we performed a mutational analysis on a prototype TEII essential for efficient production of the lipopeptide antibiotic surfactin TEIIsrf which led to identification of catalytic and structural residues. On the basis of sequence alignment of 16 TEIIs 10 single and one double mutant of highly conserved residues of TEIIsrf were constructed and biochemically investigated. We clearly identified a catalytic triad consisting of Ser86 Asp190 and His216 suggesting that TEIIsrf belongs to the a b-hydrolase superfamily. Exchange of these residues with residues with aliphatic side chains abolished enzyme activity whereas replacement of the active-site Ser86 with cysteine produced an enzyme with marginally reduced activity. In contrast exchange of the second strictly conserved asparagine Asp 163 with Ala resulted in an active but unstable enzyme excluding a role for this residue in catalysis and suggesting a structural function. The results define three catalytic and at least one structural residue in a nonribo-somal peptide synthetase TEII. Keywords catalytic triad fatty acid synthases nonribosomal peptide synthesis peptide synthetases type II thioesterase polyketide synthases. Enzymes that cleave thioesters are ubiquitous in prokaryotes and eukaryotes as thioesters appear .