title: (31)P NMR studies of O-acetylserine sulfhydrylase-B from Salmonella typhimurium.
abstract: O-Acetylserine sulfhydrylase (OASS) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the conversion of O-acetylserine and bisulfide to l-cysteine and acetate in bacteria and higher plants. Enteric bacteria have two isozymes of OASS, A and B, produced under aerobic and anaerobic growth conditions, respectively, with different substrate specificities. The (31)P chemical shift of the internal and external Schiff bases of PLP in OASS-B are further downfield compared to OASS-A, suggesting a tighter binding of the cofactor in the B-isozyme. The chemical shift of the internal Schiff base (ISB) of OASS-B is 6.2 ppm, the highest value reported for the ISB of a PLP-dependent enzyme. Considering the similarity in the binding sites of the PLP cofactor for both isozymes, torsional strain of the C5-C5' bond (O4'-C5'-C5-C4) of the Schiff base is proposed to contribute to the further downfield shift. The chemical shift of the lanthionine external Schiff base (ESB) of OASS-B is 6.0 ppm, upfield from that of unliganded OASS-B, while that of serine ESB is 6.3 ppm. Changes in chemical shift suggest the torsional strain of PLP changes as the reaction proceeds. The apoenzyme of OASS-B was prepared using hydroxylamine as the resolving reagent. Apoenzyme was reconstituted to holoenzyme by addition of PLP. Reconstitution is pseudo-first order and exhibits a final maximum recovery of 81.4%. The apoenzyme shows no visible absorbance, while the reconstituted enzyme has a UV-visible spectrum that is nearly identical to that of the holoenzyme. Steady-state fluorescence spectra gave tryptophan emission of the apoenzyme that is 3.3-fold higher than the emission of either the native or reconstituted enzyme, suggesting that PLP is a potent quencher of tryptophan emission.
authors.affiliation: Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019, USA.
authors.forename: Rong
authors.initials: R
authors.lastname: Guan
authors.forename: Susan A
authors.initials: SA
authors.lastname: Nimmo
authors.forename: Klaus D
authors.initials: KD
authors.lastname: Schnackerz
authors.forename: Paul F
authors.initials: PF
authors.lastname: Cook
mesh_terms: D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D003067:Coenzymes / Q000378:metabolism; D003547:Cysteine Synthase / Q000737:chemistry* / Q000378:metabolism; D019811:Hydroxylamine / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011732:Pyridoxal Phosphate / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology*
chemical_list: D001051:Apoenzymes; D003067:Coenzymes; D007527:Isoenzymes; D019811:Hydroxylamine; D011732:Pyridoxal Phosphate; D003547:Cysteine Synthase