Fine tuning of the active site modulates specificity in the interaction of O-acetylserine sulfhydrylase isozymes with serine acetyltransferase

Abstract

O-acetylserine sulfhydrylase (OASS) catalyzes the synthesis of l-cysteine in the last step of the reductive sulfate assimilation pathway in microorganisms. Its activity is inhibited by the interaction with serine acetyltransferase (SAT), the preceding enzyme in the metabolic pathway. Inhibition is exerted by the insertion of SAT C-terminal peptide into the OASS active site. This action is effective only on the A isozyme, the prevalent form in enteric bacteria under aerobic conditions, but not on the B-isozyme, the form expressed under anaerobic conditions. We have investigated the active site determinants that modulate the interaction specificity by comparing the binding affinity of thirteen pentapeptides, derived from the C-terminal sequences of SAT of the closely related species Haemophilus influenzae and Salmonella typhimurium, towards the corresponding OASS-A, and towards S. typhimurium OASS-B. We have found that subtle changes in protein active sites have profound effects on protein-peptide recognition. Furthermore, affinity is strongly dependent on the pentapeptide sequence, signaling the relevance of P3-P4-P5 for the strength of binding, and P1-P2 mainly for specificity. The presence of an aromatic residue at P3 results in high affinity peptides with K(diss) in the micromolar and submicromolar range, regardless of the species. An acidic residue, like aspartate at P4, further strengthens the interaction and results in the higher affinity ligand of S. typhimurium OASS-A described to date. Since OASS knocked-out bacteria exhibit a significantly decreased fitness, this investigation provides key information for the development of selective OASS inhibitors, potentially useful as novel antibiotic agents.

Isozyme-Specific Ligands for O-acetylserine sulfhydrylase, a Novel Antibiotic Target

“…In addition, as already discussed [19], G230 is substituted by R210, a residue that in some microorganisms plays a role in the selection of O -phosphoserine as the preferred substrate of the B isoform [53], [109]. The higher conservation degree of the residues belonging to the N-terminal domain with respect to those of the C-terminal domain allow the last two residues of pentapeptides docked in the active sites of OASS-A and OASS-B to occupy similar positions [19]. In spite of these common features, only OASS-A is able to interact with high affinity with SAT [55], [56].…”

“…The biochemical investigation of OASS-A and OASS-B reactivity [36] and active site specificity probed by pentapeptides [19] indicate that, despite an overall 40% sequence identity and a 70% sequence identity for the first active site shell (Figure 2A), the two isozymes exhibit subtle but significant structural differences (Figure 2B,C). Most of the residues of the first active site shell are conserved, with residues belonging to the N-terminal domain (residues 1–12 and 35–145, OASS-A numbering [47]) showing a 90% identity, residue P67 being substituted by A69 in OASS-B.…”

“…This conclusion is supported by extensive mutational and computational analysis [16], [61], also showing the relevance of the C-terminal amino acid isoleucine for OASS-SAT formation [51], [65]. The contribution of individual amino acids contained in the C-terminal sequence of SAT to complex formation and to binding specificity towards OASS-A and OASS-B was investigated using a small library of pentapeptides [19], [66]. Furthermore, recently, inhibitors for OASS-A have been obtained via a classical medicinal chemistry approach [18] and by virtual screening [11], [67].…”

“…OASS-A and OASS-B were expressed and purified as previously described [19]. The binding affinity of selected ligands to OASS-A and OASS-B was determined by monitoring the increase in fluorescence emission of the bound PLP at 500 nm following excitation at 412 nm [16], [55].…”

Moonlighting O-acetylserine sulfhydrylase: New functions for an old protein

“…Subsequent experimental measurement of peptide binding affinities for HiCysK, E. coli CysK (EcCysK) and StCysK confirms the predicted increases in binding affinity [53, 60]. However, the key energetic contribution to binding arises from an aromatic residue, specifically Tyr, at position P2, irrespective of residues at P3.…”