Prediction of substrate‐specific pockets in cyclosporin synthetase

Abstract

Amino acid sequence comparisons between domains of cyclosporin synthetase have been used to identify regions of the sequence which are responsible for the recognition and binding of the individual amino acids. Using a limited set of selection rules it was possible to identify three amino acid positions in the subdomain sequences which are responsible for amino acid specificity. Homology with the firefly luciferase protein shows that these three key residues are close to each other and line the surface of a putative specific substrate binding pocket located on the amino acyl-adenylation subdomain. These results allow us to predict a large number of cyclosporin synthetase mutants which could be used to synthesise alternative cyclosporin-like peptides.

Structure and localization of cyclosporin synthetase, the key enzyme of cyclosporin biosynthesis in Tolypocladium inflatum

“…The predicted gene product contains 11 amino-acid-activating modules that are very similar to one another and to the domains of other peptide synthetases ): each module is responsible for the recognition, activation and modification of a substrate amino acid. Seven of these modules harbor N-methyltransferase functions localized on methyltransferase domains (Husi et al 1997). Some of these features are reflected in the overall structure of the cyclosporin synthetase molecules, as depicted in Fig.…”

“…1F). Actually, as deduced from alignment procedures of the amino acid sequence, seven of the eleven modules contain three large domains, composed of 450-460 amino acids, respectively (Husi et al 1997), and representing the condensation, adenylation and methylation domains. Two smaller domains, one responsible for thioester formation (75 amino acids) and the other of unknown function (54 amino acids), must be also present but they could not be resolved in the electron microscopic image.…”

Classification of the Adenylation and Acyl-Transferase Activity of NRPS and PKS Systems Using Ensembles of Substrate Specific Hidden Markov Models

“…As mentioned before, residue selections have been made to improve the prediction. For the A domain a selection was made from the so-called core motifs [36], [39], [40], [41] and then the selection was further restricted to the active site residues [26], [27], [28]. Similarly in the case of the AT domain, the selection was at first restricted to the active site and some adjacent residues [24], [25] and later extended [31], [32].…”

Progress toward re-engineering non-ribosomal peptide synthetase proteins: a potential new source of pharmacological agents

“…In early work, preceding the report of the structure of PheA, Husi et al [1997] determined, in silico, which residues of an adenylation domain determined substrate specificity. These investigators aligned A domain sequences from cyclosporin synthetase, predicted secondary structures, and hypothesized three positions involved in conferring specificity.…”

“…Their rules for determining a specificity-conferring residue demanded that the same residue must reside at that position in domains that bind the same substrate and must be different in domains that bind different substrates. The structure of PheA was released after their work was completed, but in a note in their study, Husi et al [1997] reported that the three residues they identified are involved in forming a binding pocket for phenylalanine. Stachelhaus et al [1999] determined which residues of the A domain are responsible for substrate specificity and demonstrated their involvement by altering or relaxing substrate specificity via the mutation of one or two residues.…”