title: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Parma, Parma, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Spyrakis
authors.forename: Paolo
authors.initials: P
authors.lastname: Felici
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Bayden
authors.forename: Enea
authors.initials: E
authors.lastname: Salsi
authors.forename: Riccardo
authors.initials: R
authors.lastname: Miggiano
authors.forename: Glen E
authors.initials: GE
authors.lastname: Kellogg
authors.forename: Pietro
authors.initials: P
authors.lastname: Cozzini
authors.forename: Paul F
authors.initials: PF
authors.lastname: Cook
authors.forename: Andrea
authors.initials: A
authors.lastname: Mozzarelli
authors.forename: Barbara
authors.initials: B
authors.lastname: Campanini
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D003547:Cysteine Synthase / Q000737:chemistry* / Q000378:metabolism; D006193:Haemophilus influenzae / Q000201:enzymology*; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology*; D051050:Serine O-Acetyltransferase / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D007527:Isoenzymes; D010455:Peptides; D051050:Serine O-Acetyltransferase; D003547:Cysteine Synthase