Exploring O-acetylserine sulfhydrylase-B isoenzyme from Salmonella typhimurium by fluorescence spectroscopy

Salsi, Enea; Guan, R.; Campanini, Barbara; Bettati, Stefano; Lin, Jianling; Cook, Paul F.; Mozzarelli, Andrea

doi:10.1016/j.abb.2010.10.005

Cited by 8 publications

(10 citation statements)

References 26 publications

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“…The emission spectrum of OASS-A, upon excitation at 412 nm, was centred at 505 nm 8 , 35 and disappeared upon addition of F-Ala ( Figure 2(A) ), in agreement with the previous studies indicating that the fluorescence quantum yield of the α-aminoacrylate is much lower than that of the internal aldimine 8 . When the reaction with F-Ala was carried out on OASS-B ( Figure 2(B) ), only a shift to 550 nm of the emission peak was observed, in agreement with our previous work 7 . This emission was attributed to an α-aminoacrylate located in an active site with a different microenvironment compared to OASS-A isozyme.…”

Section: Resultssupporting

confidence: 92%

“…Changes in intensity are accompanied by a small blue shift to 501 nm that shifts back slowly to 505 nm after 7 h of incubation. The intense emitting species might be an external aldimine, because in most PLP-dependent enzymes, including OASS, the external aldimine is endowed by high fluorescence intensity 7 , 8 . The small blue shift suggests the formation of a transient species.…”

Section: Resultsmentioning

confidence: 99%

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Inhibition ofO-acetylserine sulfhydrylase by fluoroalanine derivatives

Franko

Grammatoglou

Campanini

et al. 2018

Journal of Enzyme Inhibition and Medicinal Chemistry

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O-acetylserine sulfhydrylase (OASS) is the pyridoxal 5′-phosphate dependent enzyme that catalyses the formation of L-cysteine in bacteria and plants. Its inactivation is pursued as a strategy for the identification of novel antibiotics that, targeting dispensable proteins, holds a great promise for circumventing resistance development. In the present study, we have investigated the reactivity of Salmonella enterica serovar Typhimurium OASS-A and OASS-B isozymes with fluoroalanine derivatives. Monofluoroalanine reacts with OASS-A and OASS-B forming either a stable or a metastable α-aminoacrylate Schiff’s base, respectively, as proved by spectral changes. This finding indicates that monofluoroalanine is a substrate analogue, as previously found for other beta-halogenalanine derivatives. Trifluoroalanine caused different and time-dependent absorbance and fluorescence spectral changes for the two isozymes and is associated with irreversible inhibition. The time course of enzyme inactivation was found to be characterised by a biphasic behaviour. Partially distinct inactivation mechanisms for OASS-A and OASS-B are proposed.

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Section: Resultssupporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Inhibition ofO-acetylserine sulfhydrylase by fluoroalanine derivatives

Franko

Grammatoglou

Campanini

et al. 2018

Journal of Enzyme Inhibition and Medicinal Chemistry

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“…The resulting complex eluted with an estimated molecular mass of 476 kDa, which is substantially greater than the expected mass of 336 kDa for the CSC. To determine the stoichiometry of the complex, we performed titrations to monitor changes in the fluorescent emission from the PLP cofactor of CysK as a function of CysE concentration (Fig. B).…”

Section: Resultsmentioning

confidence: 99%

“…CysK from E. coli and CysM from S. Typhimurium were expressed recombinantly in E. coli BL21(DE3) and purified by ion metal affinity chromatography (IMAC) on immobilized Co 2+ ions (Talon Technology, Clontech Laboratories, Inc., Mountain View, CA, USA), following with minor modifications. His‐tag was removed from StCysM by incubation at 37 °C using Factor Xa in a 1 : 200 ratio with protein in 20 m m Hepes, 100 m m NaCl, and 4 m m CaCl 2 , pH 7.5.…”

Section: Methodsmentioning

confidence: 99%

“…The second reaction is catalyzed by O ‐acetylserine sulfhydrylase‐A (CysK), a pyridoxal 5′‐phosphate (PLP)‐dependent enzyme that displaces the acetoxy group from O ‐acetylserine with bisulfide to yield l‐ Cys . Many bacteria also encode O ‐acetylserine sulfhydrylase‐B (CysM) that is thought to play an important role in l‐ Cys biosynthesis under stress conditions .…”

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confidence: 99%

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Modulation of Escherichia coli serine acetyltransferase catalytic activity in the cysteine synthase complex

Benoni

Bei

Paredi³

et al. 2017

FEBS Letters

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In bacteria and plants, serine acetyltransferase (CysE) and O-acetylserine sulfhydrylase-A sulfhydrylase (CysK) collaborate to synthesize L-Cys from L-Ser. CysE and CysK bind one another with high affinity to form the cysteine synthase complex (CSC). We demonstrate that bacterial CysE is activated when bound to CysK. CysE activation results from the release of substrate inhibition, with the K i for L-Ser increasing from 4 mM for free CysE to 16 mM for the CSC. Feedback inhibition of CysE by L-Cys is also relieved in the bacterial CSC. These findings suggest that the CysE active site is allosterically altered by CysK to alleviate substrate and feedback inhibition in the context of the CSC. Author contributions BC, SB, and AM conceived and supervised the study; RB, ODB, GP, and NF performed experiments; CSH provided the expression vectors; BC, RB, and ODB analyzed the data; BC prepared the original draft; BC, SB, CSH, and AM reviewed and edited the manuscript. Supporting informationAdditional Supporting Information may be found online in the supporting information tab for this article. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptPlants and bacteria share a common two-reaction pathway for the synthesis of L-cysteine (LCys) from L-serine (L-Ser; Fig. 1). Serine acetyltransferase (CysE) catalyzes an acyl transfer from acetyl-CoA to L-Ser using a random-order kinetic mechanism [1]. The second reaction is catalyzed by O-acetylserine sulfhydrylase-A (CysK), a pyridoxal 5′-phosphate (PLP)-dependent enzyme that displaces the acetoxy group from O-acetylserine with bisulfide to yield L-Cys [2][3][4][5][6][7][8]. Many bacteria also encode O-acetylserine sulfhydrylase-B (CysM) [9,10] that is thought to play an important role in L-Cys biosynthesis under stress conditions [11].Kredich et al. [2,12] first discovered that CysE and CysK from Salmonella Typhimurium bind to one another with high affinity, and they called this assembly the cysteine synthase complex (CSC; Fig. 1). The CysE-CysK interaction is highly conserved across species, and the plant enzymes also form a high-affinity CSC. Although there is no experimentally solved structure available for the CSC, biochemical and spectroscopic approaches revealed that the C-terminal tail of CysE inserts into the CysK active site to anchor the interaction. CysE proteins that lack C-terminal residues are unable to bind CysK [13][14][15], and CSC formation is disrupted by millimolar O-acetylserine, which competes with CysE for binding to the CysK active site [12,16,17]. These findings are supported by crystal structures of CysE Cterminal peptides bound in the active site of CysK. These structures show that the C-terminal Ile residue of CysE engages in the same specific interactions with the active site as Oacetylserine substrate [18,19]. The stoichiometry of CysE to CysK has been determined to be 3:2 for CSCs from S. Typhimurium and Haemophilus influenzae. Because CysK forms homodimers and CysE exists as a dimer of trimers [20,21],...

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