1. Di Vincenzo L, Grgurina I, Pascarella S
Computational analysis of structural properties of classical and novel non ribosomal aminoacyladenylate forming domains.
Meeting: BITS 2004 - Year: 2004
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Abstract: Nonribosomal peptide synthetases (NRPSs) are multidomain, multifunctional enzymes involved in the biosynthesis of many bioactive microbial peptides such as phytotoxins, siderophores, biosurfactants, and anticancer agents. The minimal module required for a single monomer addition consists of a condensation domain (C), an adenylation domain (A) and a peptidyl carrier protein (PCP) domain also denoted as thiolation (T) domain. Systematic comparative analyses identified 8 or 10 sequence positions lining the active site pocket which are held responsible for substrate recognition and selection in A domain. Recently, it has been pointed out that several enzymes possibly involved in lysine metabolism in eucaryotes display a 3-domain architecture where the two N-terminal domains are homologous to the A and T domains from NRPS systems. The third C-terminal section may contain a PQQ, a NADPH or a functionally uncharacterized domain. Our work is aimed at the structural characterization and the study of common molecular features of the family of the aminoacyladenylate-forming enzymes from NRPS and from the recently discovered homologous enzymes. Psi-BLAST searches were applied over the GeAll and Non-Redundant databanks using query sequences Ebony (gi:3286766) from Drosophila melanogaster, 5-aminoadipic acid synthase (gi:30348962) from Mus musculus and aminoadipate-semialdehyde dehydrogenase from yeast (swissprot:LYS2_YEAST). Thirty-two sequences were identified from different eucaryotic species and the domain assignments were confirmed by CDD and Pfam queries. The sequence subsets containing the A-T domains were aligned utilizing the HMMER package. On the basis of the structural homology encoded in this multiple alignment, the potential occurrence of a “specificity code” similar to that described for the NRPS systems has been tested. The residues which interact with the α-amino and α-carboxy groups of the amino acid substrates , Asp235 and Lys517 respectively, are conserved, the only exceptions being Ebony protein (gi:3286766) from Drosophila melanogaster and (gi:21291643) from Anopheles gambiae where the Asp235 is replaced by valine. Homology molecular modelling has been utilized to map the conserved residues onto a hypothetical active site structure of the 5-aminoadipic acid synthase from Homo sapiens (gi:32261239) and Ebony (gi:3286766) from Drosophila melanogaster to understand the role of the conserved residues and to predict their interaction with the putative substrates. In case of Ebony proteins, the Asp235 is replaced by Val, while Pro236, conserved in all 5-aminoadipic acid synthase and aminoadipate-semialdehyde dehydrogenase, is substituted by Asp which can form hydrogen bond with the β-amino group of the β-alanine substrate. The β-amino group interacts via hydrogen bonds also with Ser301 and Asp331. The other residues line and shape the active site pocket. Characterization of the α-aminoadipate synthase is under way.