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Substrate Recognition and Selection by the Initiation Module PheATE of Gramicidin S Synthetase

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    Substrate Recognition and Selection by the Initiation Module PheATE of Gramicidin S Synthetase
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    Contribution from the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115
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    Journal of the American Chemical Society

    Cite this: J. Am. Chem. Soc. 2001, 123, 45, 11208–11218
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    https://doi.org/10.1021/ja0166646
    Published October 11, 2001
    Copyright © 2001 American Chemical Society
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    Abstract

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    The initiation module of non-ribosomal peptide synthetases (NRPS) selects and activates the first amino acid and serves as the aminoacyl donor in the first peptide bond-forming step of the NRPS assembly line. The gramicidin S synthetase initiation module (PheATE) is a three-domain subunit, recognizing l-phenylalanine (l-Phe) and activating it (by adenylation domain) as tightly bound l-phenylalanyl-adenosine-5‘-monophosphate diester (l-Phe-AMP), transferring it to the HS-phosphopantetheine arm of the holo-thiolation (holo-T) domain, and then epimerizing it (by epimerization domain) to the d-Phe-S-4‘-Ppant-acyl enzyme. In this study, we have assayed the selectivity of the PheATE adenylation domain with a number of proteinogenic amino acids and observed that three additional amino acids, l-Tyr, l-Trp, and l-Leu, were activated to the aminoacyl-AMPs and transferred to the HS-phosphopantetheine arm of the holo-T domain. Hydrolytic editing of noncognate aminoacyl-AMPs and/or aminoacyl-S-4‘-Ppant-acyl enzymes by the enzyme was not observed by three different assays for adenylation domain function. The microscopic reaction rates and thermodynamic equilibrium constants obtained from single-turnover studies of reactions of l-Phe, l-Trp, l-Tyr, and l-Leu with holoPheATE allowed us to construct free energy profiles for the reactions, revealing the kinetic and thermodynamic basis for substrate recognition and selection. In particular, the rates of epimerization of the l-aminoacyl-S-enzyme to the d-aminoacyl-S-enzyme intermediate showed reductions of 245-, 300-, and 540-fold for l-Trp, l-Tyr, and l-Leu respectively, suggesting that the epimerization domain is an important gatekeeper for generation of the d-Phe-S-enzyme that starts gramicidin S chain growth.

    Copyright © 2001 American Chemical Society

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    †

     Present address:  Institute of Biochemistry, Department of Chemistry, Philipps-University Marburg, Hans-Meerwein-Str., 35032 Marburg, Germany.

    *

     To whom correspondence should be addressed. Phone:  (617) 432 1715. Fax:  (617) 432 0438. E-mail:  [email protected].

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    Cite this: J. Am. Chem. Soc. 2001, 123, 45, 11208–11218
    Click to copy citationCitation copied!
    https://doi.org/10.1021/ja0166646
    Published October 11, 2001
    Copyright © 2001 American Chemical Society
    Request reuse permissions

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