An active serine is involved in covalent substrate amino acid binding at each reaction center of gramicidin S synthetase

• Published in: The Journal of biological chemistry 1991-12, Vol.266 (34), p.23135-23141
• Main Authors: SCHLUMBOHM, W, STEIN, T, ULLRICH, C, VATER, J, KRAUSE, M, MARAHIEL, M. A, KRUFT, V, WITTMANN-LIEBOLD, B
• Format: Article
• Language: English
• Subjects: Amino Acid Isomerases - chemistry; Amino Acid Isomerases - metabolism; Amino Acid Sequence; Amino Acids - metabolism; Analytical, structural and metabolic biochemistry; Binding Sites; Biological and medical sciences; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; gramicidin S synthase; Leucine - chemistry; Miscellaneous; Molecular Sequence Data; Multienzyme Complexes - chemistry; Multienzyme Complexes - metabolism; Peptide Synthases - chemistry; Peptide Synthases - metabolism; Sequence Alignment; Serine - chemistry; Serine - metabolism; Substrate Specificity; Valine - chemistry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)54473-2

The condensing peptide forming multienzyme of gramicidin S synthetase (gramicidin S synthetase 2) was specifically labeled at its putative thiotemplate sites for L-valine and L-leucine by covalent incorporation of the 14C-labeled substrate amino acids. The thioester complexes of the multienzyme were digested with CNBr, Staphylococcus aureus V8 protease, and pepsin. Reaction center peptides containing the [14C]valine and [14C]leucine labels were isolated in pure form. They show a high degree of sequence similarity and contain the same consensus sequence LGGH/DXL. The labels were eliminated in the first Edman degradation step. A dehydroalanine was identified which can originate from either a cysteine or a serine. The comparison of the chemical results with the deduced amino acid sequence of the grsB gene encoding the gramicidin S synthetase 2 revealed that 4 such motifs are located within the gene structure, each of them being localized in the 3'-terminal region of one of 4 gene segments grsB1-B4. They have a size of approximately 2 kilobases and presumably code for the 4 amino acid activating domains of the synthetase. Surprisingly a serine was found at each putative substrate amino acid-binding position instead of a cysteine as postulated by the thiotemplate mechanism. Therefore the data suggest that active serine residues are involved in nonribosomal peptide syntheses of microbial peptides.