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 [2], 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.

Abstract: This work presents a computational analysis of the molecular characteristics shared by the A domains from traditional nonribosomal peptide synthetases (NRPSs) and the group of the freestanding homologous enzymes: a-aminoadip ate semialdehyde dehydrogenase, a- aminoadipate reductase and the protein Ebony.

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Abstract: Serine hydroxymethyltransferase (SHMT: E.C. 2.1.2.1) catalyzes the reversible conversion of serine and tetrahydropteroylglutamate (H4PteGlu) to glycine and 5,10-methylene-H4PteGlu. This reaction is the major source of one-carbon groups required in the biosynthesis of methionine, choline, thymidylate, and purines. The SHMT enzyme is widely distributed in nature, and found in both prokaryotic and eukaryotic cells with the latter containing both cytosolic and mitochondrial forms. In addition to H4PteGlu, the enzyme also requires pyridoxal 5'-phosphate (PLP) as a coenzyme. It is one of a group of PLP enzymes that cleave one of the bonds at the a-carbon of their amino acid substrate. These are referred to as the _-family of PLP enzymes and include the transaminases and amino acid decarboxylases. Three-dimensional structures have been determined for several members of the _-family of vitamin B6-dependent enzymes. Amongst them are aspartate aminotransferase (AAT), tyrosine phenol-lyase (1TPL), _-amino acid aminotransferase, 2,2-dialkylglycine decarboxylase (2DKB), ornithine aminotransferase, glutamate-1-semialdehyde aminomutase, and ornithine decarboxylase (1ORD). Despite the widely differing reaction specificity and weak sequence similarity, these proteins share the same basic folding pattern which was assumed also for SHMT (2). The prolonged unavailability of the SHMT atomic structure prompted the construction of a three-dimensional "homology" model that integrated knowledge regarding the enzyme acquired through site-directed mutagenesis experiments and other experimental measures. E. coli SHMT was modelled because most of experimental data are available for this enzyme. New features of the active site are proposed for further experimental testing.

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