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 production of the lipopeptide antibiotic surfactin (TEII(srf)), 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 TEII(srf) were constructed and biochemically investigated. We clearly identified a catalytic triad consisting of Ser86, Asp190 and His216, suggesting that TEII(srf) belongs to the alpha/beta-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 (Asp163) 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 nonribosomal peptide synthetase TEII.
Linne
Uwe
U
Philipps Universität Marburg, Fachbereich Chemie/Biochemie, Hans-Meerwein-Strassse, 35032 Marburg, Germany.
Schwarzer
Dirk
D
Schroeder
Gunnar N
GN
Marahiel
Mohamed A
MA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D017124:Conserved Sequence; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D064429:Fatty Acid Synthases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055666:Lipopeptides; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009876:Operon / Q000235:genetics; D010453:Peptide Synthases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010456:Peptides, Cyclic / Q000378:metabolism; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013869:Thiolester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D055666:Lipopeptides; D010456:Peptides, Cyclic; D011994:Recombinant Proteins; C009365:surfactin peptide; D004228:Dithionitrobenzoic Acid; D064429:Fatty Acid Synthases; D006867:Hydrolases; D013869:Thiolester Hydrolases; C022738:thioesterase II; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase