Structurally conserved regions and hydrophobic contacts in the fold type I, PLP-dependent enzymes

INTRODUCTION: The interacting hydrophobic residues conserved at primary and tertiary structure levels have been investigated in the case of fold-type I, pyridoxal-5’- phosphate (PLP) dependent enzymes. Although there are at least five evolutionarily unrelated superfamilies of PLP-dependent enzymes, each displaying a completely different fold, the by far largest and best characterised is known as fold-type I, family, or aspartate aminotransferase family. This large group of enzymes, that are found in all organisms and cover together the whole range of enzymatic activities catalogued by the Enzyme Commission, bears several interesting characteristics: its members are highly divergent enzymes which display structural homology with almost undetectable sequence similarity; thanks to the recent massive sequencing of several genomes and advances in protein structure determination, a good wealth of experimentally well characterised information is now available for this superfamily. On the basis of such consideration, the present work was aimed at detecting the conserved structural patterns possibly responsible for the maintenance of the fold of this protein superfamily. The role played by conserved residues in the stabilisation of the native structure and their possible involvement in the mechanism of protein folding was then discussed in the light of the most recent studies on PLP-dependent enzymes. MATERIAL AND METHODS: a non redundant set of 23 structures of PLP dependent enzymes of type I was collected from the databanks. An initial structural alignment was obtained with the program CE (1) and was subsequently refined by hand. Structurally Conserved Regions (SCR) were defined as those regions displaying similar local conformation (RMSD over superposed Cα atoms less than 3.0 Å), with no indel and at least three-residue long. Pairwise hydrophobic contact areas were calculated for every possible residue belonging to SCRs. The equivalent hydrophobic contacts present in most of the superposed structures were defined as Conserved Hydrophobic Contacts (CHC). Sequence homologs were collected from the databanks and, after a check for redundancy, aligned to the structural alignment for a total of 921 sequences. RESULTS: Most of the sequences collected and aligned display pairwise sequence identity in the range 0%-20% which encompass the “twilight zone”. This suggest that the data set is suitable to explore distant evolutionary events. Fifteen CHCs involving 22 residues were collected at the end of the analysis. These CHCs can be grouped into three clusters: one is located in the proximity of the PLP molecule, the second in the minor domain, and the third in the hinge region between two SCRs (Figure 1). Seventeen out of the 22 residues involved in CHCs are highly conserved across the multiple alignment of 921 sequences. Sequence conservation whithin the SCRs has also been analysed. Eleven sites within the SCRs not involved in CHCs display a significant sequence conservation. These position can be grouped in two categories: i) Gly or Ala rich positions in the proximity of CHC residues and ii) positions involved in hydrophobic contacts that do not meet the criteria followed to define a significant CHC. The CHCs and the conserved positions within the SCRs suggest the existence of determinants necessary to the stabilization and the maintaining of the fold of the PLP type I enzymes. We suggest that this analysis can be extended to other families and can help planning experiments of protein folding and design.