Analysis of core sequences in the D-Phe activating domain of the multifunctional peptide synthetase TycA by site-directed mutagenesis.
The D-phenylalanine-activating enzyme tyrocidine synthetase I (TycA) from Bacillus brevis ATCC 8185 was overexpressed in Escherichia coli, purified to homogeneity, and assayed for ATP-PPi exchange and covalent binding of phenylalanine by the thiotemplate mechanism. Amino acid exchanges in four different cores of TycA created by site-directed mutagenesis revealed the amino acid residues involved in aminoacyladenylate formation and in covalent thioester formation. Mutations in the putative ATP-binding site SGTTGKPKG caused a decreased phenylalanine-dependent ATP-PPi exchange activity to 10% of the wild-type level for a Lys-186-to-Arg substitution and an almost complete loss of activity (< 1%) for a Lys-186-to-Thr exchange. Alteration of Asp-401 to Asn in the ATPase motif TGDL of TycA decreased the phenylalanine-dependent ATP-PPi exchange activity to 75% of wild type, while an Asp-401-to-Ser mutation decreased the activity to 10% of the wild-type level. Replacement of Ser-562 in the putative thioester-binding motif LGGDSI to Ala or Gly caused a reduction in trichloroacetic acid-precipitable TycA-[14C]phenylalanine complex to one-third of the wild-type level. However, no cleavable [14C]phenylalanine could be detected after treatment with performic acid, indicating that the resulting mutant was unable to form thioester with phenylalanine. In E. coli, TycA was labeled with beta-[3H]alanine, a precursor of 4'-phosphopantetheine, indicating that TycA is modified with a beta-alanine-containing cofactor.
Gocht
M
M
Fachbereich Chemie/Biochemie, Philipps-Universität Marburg, Germany.
Marahiel
M A
MA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D011756:Diphosphates / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010453:Peptide Synthases / Q000235:genetics / Q000378:metabolism*; D010649:Phenylalanine / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D013237:Stereoisomerism; D015091:beta-Alanine / Q000378:metabolism
D011756:Diphosphates; D011994:Recombinant Proteins; D015091:beta-Alanine; D010649:Phenylalanine; D000255:Adenosine Triphosphate; D010453:Peptide Synthases; C035180:tyrocidine synthetase