Portability of epimerization domain and role of peptidyl carrier protein on epimerization activity in nonribosomal peptide synthetases.

Abstract:

:Incorporation of nonproteinogenic amino acids in small polypeptides synthesized by nonribosomal peptide synthetases (NRPS) significantly contributes to their biological activity. In these peptides, conversion of L-amino acids to the corresponding D-isomer is catalyzed by specialized NRPS modules that utilize an epimerization (E) domain. To understand the basis for the specific interaction of E domains with PCP domains (peptidyl carrier proteins, also described as T domains) and to investigate their substrate tolerance, we constructed a set of eight fusion proteins. The gene fragments encoding E and PCP-E domains of TycA (A-PCP-E), the one module tyrocidine synthetase A, were fused to different gene fragments encoding A and A-PCP domains, resulting in A/PCP-E and A-PCP/E types of fusion proteins (slash indicates site of fusion). We were able to show that the E domain of TycA, usually epimerizing Phe, does also accept the alternate substrates Trp, Ile, and Val, although with reduced efficiency. Interestingly, however, an epimerization activity was only observed in the case of fusion proteins where the PCP domain originates from modules containing an E domain. Sequence comparison revealed that such PCPs possess significant differences in the signature Ppant binding motif (CoreT: [GGDSI]), when compared to those carrier proteins, originating from ordinary C-A-PCP elongation modules (CoreT: [GGHSL]). By means of mutational analysis, we could show that epimerization activity is influenced by the nature of amino acid residues in proximity to the cofactor Ppant binding site. The aspartate residue in front of the invariant serine (Ppant binding site) especially seems to play an important role for the proper interaction between PCP and the E domain, as well as the presentation of the aminoacyl-S-Ppant substrate in the course of substrate epimerization. In conclusion, specialized PCP domains are needed for a productive interaction with E domains when constructing hybrid enzymes.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Linne U,Doekel S,Marahiel MA

doi

10.1021/bi011595t

subject

Has Abstract

pub_date

2001-12-25 00:00:00

pages

15824-34

issue

51

eissn

0006-2960

issn

1520-4995

pii

bi011595t

journal_volume

40

pub_type

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