Carrier Protein Recognition in Siderophore-Producing Nonribosomal Peptide Synthetases

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• 作者：C. Gary Marshall ; Michael D. Burkart ; Robin K. Meray ; and Christopher T. Walsh
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：July 2, 2002
• 年：2002
• 卷：41
• 期：26
• 页码：8429 - 8437
• 全文大小：135K
• 年卷期：v.41,no.26(July 2, 2002)
• ISSN：1520-4995

文摘

Nonribosomal peptide synthetases (NRPSs) use phosphopantetheine (pPant) bearing carrierproteins to chaperone activated aminoacyl and peptidyl intermediates to the various enzymes that effectpeptide synthesis. Using components from siderophore NRPSs that synthesize vibriobactin, enterobactin,yersiniabactin, pyochelin, and anguibactin, we examined the nature of the interaction of such cofactor-carrier proteins with acyl-activating adenylation (A) domains. While VibE, EntE, and PchD were all ableto utilize "carrier protein-free" pPant derivatives, the pattern of usage indicated diversity in the bindingmechanism, and even the best substrates were down at least 3 log units relative to the native cofactor-carrier protein. When tested with four noncognate carrier proteins, EntE and VibE differed both in therange of substrate utilization efficiency and in the distribution of the efficiencies across this range.Correlating sequence alignments to kinetic efficiency allowed for the construction of eight point mutantsof VibE's worst substrate, HMWP2 ArCP, to the corresponding residue in its native VibB. Mutants S49Dand H66E combined to increase activity 6.2-fold and had similar enhancing effects on the downstreamcondensation domain VibH, indicating that the two NRPS enzymes share carrier protein recognitiondeterminants. Similar mutations of HMWP2 ArCP toward EntB had little effect on EntE, suggesting thatthe position of recognition determinants varies across NRPS systems.