Nonribosomal peptide synthetases-evidence for a second ATP-binding site

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• 作者：Kallow ; Wibke ; Pavela-Vrancic ; Maja ; Dieckmann ; Ralf ; von Dö ; hren ; Hans
• 关键词：ACV synthetase ; Aminoacyl adenylation ; Substrate inhibition ; NRPS ; nonribosomal peptide synthetases ; ACVS ; δ ; -(l-α ; -aminoadipyl)-l-cysteinyl-d-valine synthetase ; ACV ; δ ; -(l-α ; -aminoadipyl)-l-cysteinyl-d-valine ; apo-TY1 ; tyrocidine syntheta
• 刊名：Biochimica et Biophysica Acta - Proteins and Proteomics
• 出版年：2002
• 出版时间：November 19, 2002
• 年：2002
• 卷：1601
• 期：1
• 页码：93-99
• 全文大小：188 K

文摘

δ-(l-α-Aminoadipyl)-l-cysteinyl-d-valine synthetase (ACVS) catalyses, via the protein thiotemplate mechanism, the nonribosomal biosynthesis of the penicillin and cephalosporin precursor tripeptide δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine (ACV). The complete and fully saturated biosynthetic system approaches maximum rate of product generation with increasing ATP concentration. Nonproductive adenylation of ACVS, monitored utilising the ATP–[³²P]PP_i exchange reaction, has revealed substrate inhibition with ATP. The kinetic inhibition pattern provides evidence for the existence of a second nucleotide-binding site with possible implication in the regulatory mechanism. Under suboptimal reaction conditions, in the presence of MgATP²⁻, l-Cys and inorganic pyrophosphatase, ACVS forms adenosine(5′)tetraphospho(5′)adenosine (Ap₄A) from the reverse reaction of adenylate formation involving a second ATP molecule. The potential location of the second ATP binding site was deduced from sequence comparisons and molecular visualisation in conjunction to data obtained from biochemical analysis.