Mutational analysis of the C-domain in nonribosomal peptide synthesis.
The initial condensation event in the nonribosomal biosynthesis of the peptide antibiotics gramicidin S and tyrocidine A takes place between a phenylalanine activating racemase GrsA/TycA and the first proline-activating module of GrsB/TycB. Recently we established a minimal in vitro model system for NRPS with recombinant His6-tagged GrsA (GrsAPhe-ATE; 127 kDa) and TycB1 (TycB1Pro-CAT; 120 kDa) and demonstrated the catalytic function of the C-domain in TycB1Pro-CAT to form a peptide bond between phenylalanine and proline during diketopiperazine formation (DKP). In this work we took advantage of this system to identify catalytically important residues in the C-domain of TycB1Pro-CAT using site-directed mutagenesis and peptide mapping. Mutations in TycB1Pro-CAT of 10 strictly conserved residues among 80 other C-domains with potential catalytic function, revealed that only R62A, H147R and D151N are impaired in peptide-bond formation. All other mutations led to either unaffected (Q19A, C154A/S, Y166F/W and R284A) or insoluble proteins (H146A, R67A and W202L). Although 100 nm of the serine protease inhibitors N-alpha-tosyl-l-phenylalanylchloromethane or phenylmethanesulfonyl fluoride completely abolished DKP synthesis, no covalently bound inhibitor derivatives in the C-domain could be identified by peptide mapping using HPLC-MS. Though the results do not reveal a particular mechanism for the C-domain, they exhibit a possible way of catalysis analogous to the functionally related enzymes chloramphenicol acetyltransferase and dihydrolipoyl transacetylase. Based on this, we propose a mechanism in which one catalytic residue (H147) and two other structural residues (R62 and D151) are involved in amino-acid condensation.
Bergendahl
Veit
V
Biochemie/Fachbereich Chemie, Philipps-Universität Marburg, Germany.
Linne
Uwe
U
Marahiel
Mohamed A
MA
D000591:Amino Acid Isomerases / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D017931:DNA Primers; D006096:Gramicidin / Q000096:biosynthesis* / Q000737:chemistry; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010453:Peptide Synthases / Q000378:metabolism; D014440:Tyrocidine / Q000096:biosynthesis* / Q000737:chemistry
D017931:DNA Primers; D014440:Tyrocidine; D006096:Gramicidin; D000591:Amino Acid Isomerases; C022343:phenylalanine racemase (ATP-hydrolyzing); D010453:Peptide Synthases; C035180:tyrocidine synthetase