An active serine is involved in covalent substrate amino acid binding at each reaction center of gramicidin S synthetase.
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.
Schlumbohm
W
W
Institute of Biochemistry and Molecular Biology, Technical University of Berlin, Federal Republic of Germany.
Stein
T
T
Ullrich
C
C
Vater
J
J
Krause
M
M
Marahiel
M A
MA
Kruft
V
V
Wittmann-Liebold
B
B
D000591:Amino Acid Isomerases / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D001665:Binding Sites; D007930:Leucine / Q000737:chemistry; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism; D010453:Peptide Synthases / Q000737:chemistry* / Q000378:metabolism; D016415:Sequence Alignment; D012694:Serine / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D014633:Valine / Q000737:chemistry
D000596:Amino Acids; D009097:Multienzyme Complexes; D012694:Serine; D000591:Amino Acid Isomerases; D010453:Peptide Synthases; C020831:gramicidin-S-synthetase 2; D007930:Leucine; D014633:Valine