title: Peptide Bond Synthesis by a Mechanism Involving an Enzymatic Reaction and a Subsequent Chemical Reaction.
abstract: We recently reported that an amide bond is unexpectedly formed by an acyl-CoA synthetase (which catalyzes the formation of a carbon-sulfur bond) when a suitable acid and l-cysteine are used as substrates. DltA, which is homologous to the adenylation domain of nonribosomal peptide synthetase, belongs to the same superfamily of adenylate-forming enzymes, which includes many kinds of enzymes, including the acyl-CoA synthetases. Here, we demonstrate that DltA synthesizes not only N-(d-alanyl)-l-cysteine (a dipeptide) but also various oligopeptides. We propose that this enzyme catalyzes peptide synthesis by the following unprecedented mechanism: (i) the formation of S-acyl-l-cysteine as an intermediate via its "enzymatic activity" and (ii) subsequent "chemical" S → N acyl transfer in the intermediate, resulting in peptide formation. Step ii is identical to the corresponding reaction in native chemical ligation, a method of chemical peptide synthesis, whereas step i is not. To the best of our knowledge, our discovery of this peptide synthesis mechanism involving an enzymatic reaction and a subsequent chemical reaction is the first such one to be reported. This new process yields peptides without the use of a thioesterified fragment, which is required in native chemical ligation. Together with these findings, the same mechanism-dependent formation of N-acyl compounds by other members of the above-mentioned superfamily demonstrated that all members most likely form peptide/amide compounds by using this novel mechanism. Each member enzyme acts on a specific substrate; thus, not only the corresponding peptides but also new types of amide compounds can be formed.
authors.affiliation: From the Institute of Applied Biochemistry and Graduate School of Life and Environmental Sciences, The University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572 and; the Division of Life Science and Engineering, School of Science and Engineering, Tokyo Denki University, Hatoyama, Hiki-gun, Saitama 350-0394, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Abe
authors.affiliation: From the Institute of Applied Biochemistry and Graduate School of Life and Environmental Sciences, The University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572 and.
authors.forename: Yoshiteru
authors.initials: Y
authors.lastname: Hashimoto
authors.affiliation: From the Institute of Applied Biochemistry and Graduate School of Life and Environmental Sciences, The University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572 and.
authors.forename: Ye
authors.initials: Y
authors.lastname: Zhuang
authors.affiliation: From the Institute of Applied Biochemistry and Graduate School of Life and Environmental Sciences, The University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572 and.
authors.forename: Yin
authors.initials: Y
authors.lastname: Ge
authors.affiliation: From the Institute of Applied Biochemistry and Graduate School of Life and Environmental Sciences, The University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572 and.
authors.forename: Takuto
authors.initials: T
authors.lastname: Kumano
authors.affiliation: From the Institute of Applied Biochemistry and Graduate School of Life and Environmental Sciences, The University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572 and. Electronic address: kobay@agbi.tsukuba.ac.jp.
authors.forename: Michihiko
authors.initials: M
authors.lastname: Kobayashi
mesh_terms: D001412:Bacillus subtilis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D055162:Biocatalysis; D019729:Carbon-Oxygen Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D010455:Peptides; D019729:Carbon-Oxygen Ligases; C502532:DltA protein, Bacillus subtilis
keywords: ATP; Bacillus; adenylate-forming superfamily; amide; amino acid; enzyme mechanism; native chemical ligation; peptide biosynthesis; peptides; protein domain