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Identification of Remote Homologues of the Beta-Lactamase/DD-Peptidase Fold.

Lorenzo Segovia

DRMB- Instituto de Biotecnologia - UNAM - Cuernavaca, Mexico.

Beta-lactamases (Bls), the enzymes that hydrolyze penicillin, have been shown to be structurally related to DD-peptidases, enzymes involved in bacterial cell wall biosynthesis and natural targets of the beta-lactam antibiotics. These enzymes are also called penicillin-binding-proteins (PBPs). Their structural similarity and the conservation of certain amino acid motifs have led several authors to postulate that Bls have evolved from PBPs.

We used the amino acid sequences of those Bls and PBPs whose tertiary structures have been determined to look for homologues in database searches using BLAST and FastA. These searches give a large amount of hits, although some with marginal scores (BLAST scores and FastA scores >= 50), with enzymes not previously known to be homologues. Representatives of each class of enzyme were selected and subsequent database searches were carried out using these sequences. Only those enzymes which recognized each other within the previous parameters were kept, potentially forming a closed cluster of homology. These enzymes were found to have very diverse catalytic activities (hydrolases, transferases and esterases) acting on very diverse substrates. Noteworthy, eukaryotic homologues were found. Pair wise comparisons using the GAP algorithm and the PRDF program with 500 shuffles were carried out for all possible pairs. Some of the comparisons show homologies falling into the "twilight zone" (>20% aa. id.) and found not to be significative with PRDF (scores well within the random score distribution).

Motif searches on these sequences show the conservation of a discrete signature (SXXK) which covers some of the Bls/PBPs catalytic residues. Some of the homologues have been shown experimentally to have a catalytic S.

To validate these remote homologies, fold recognition (threading) was performed using ProFIT and the MBI-UCLA fold recognition server. In all cases the Beta-lactamase/DD-peptidase fold was the best hit.

These results and their evolutionary implications in the light of the catalytic variability of a single fold will be discussed.

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