Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UMLS:C1832526 (PCC)
 5,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cysteine desulfurases (CDs) are pyridoxal-5'-phosphate (PLP)-dependent enzymes that cleave sulfur from cysteine via an enzyme cysteinyl persulfide intermediate. In vitro studies of these enzymes have generally employed dithiothreitol as a cosubstrate to reductively cleave the persulfide intermediate, and it has been suggested that persulfide cleavage is the rate-limiting step for catalysis. In this study, the kinetics and mechanisms of cleavage of the persulfide intermediate in Slr0387 (CD-0387), a sequence group I (NifS/IscS-like) cysteine desulfurase from Synechocystis sp. PCC 6803, by physiological and nonphysiological reductants have been examined, and the extent to which this step is rate-limiting for catalysis has been determined. The observations that dithiols such as dithiothreitol (DTT) cleave the persulfide with approximately 100-fold greater efficiency than structurally similar monothiols such as 2-mercaptoethanol (2-ME), that cleavage by DTT exhibits saturation kinetics, and that the dependence of the observed first-order rate constant for persulfide cleavage by DTT on the concentration of the dithiol corresponds precisely with that for formation of a complex between DTT and the PLP cofactor of the resting enzyme suggest that persulfide cleavage by dithiols occurs by prior formation of a complex, in which addition of one thiol to the cofactor positions the second thiol for attack. This conclusion and the observation that a second molecule of L-cysteine can bind to the cofactor in the persulfide form of CD-0387 explain why several CDs are subject to potent inhibition by L-cysteine during turnover with DTT: binding of L-cysteine prevents formation of the PLP-DTT adduct and renders the dithiol no better than a monothiol, which must react with the persulfide in bimolecular fashion. Consistent with this rationale, catalysis by CD-0387 with 2-ME as cosubstrate, while less efficient, is not subject to potent inhibition by L-cysteine. The similarity of the maximum rate constant for persulfide cleavage by DTT to k(cat) suggests that persulfide cleavage is, in fact, primarily rate-determining, and this conclusion is confirmed by the observation that k(cat) is approximately 10-fold greater when tris-(2-carboxyethyl)phosphine (TCEP), the most efficient persulfide cleaver identified, is used as the reducing cosubstrate. The faster turnover with TCEP provides a chemical model for activation of CD-0387 and other CDs by the presence of accessory factors that serve as efficient acceptors of the persulfide sulfur.
 ...
PMID:Mechanism of cysteine desulfurase Slr0387 from Synechocystis sp. PCC 6803: kinetic analysis of cleavage of the persulfide intermediate by chemical reductants. 1537 60

Stopped-flow absorption and isotope effect experiments have been used to dissect the mechanism of formation of the enzyme cysteinyl persulfide intermediate in the reaction of a cysteine desulfurase (CD), CD0387 from Synechocystis sp. strain PCC 6803. Seven accumulating intermediates have been identified and tentatively mapped onto the CD chemical mechanism originally proposed by Dean, White, and co-workers [Zheng, L., White, R. H., Cash, V. L., and Dean, D. R. (1994) Biochemistry 33, 4714-4720]. The first intermediate with lambda(max) approximately 350 nm is assigned as either a gem-diamine complex or a thiol adduct formed by nucleophilic attack of either the amine group or the sulfhydryl group of the substrate on the internal aldimine form of the pyridoxal 5'-phosphate (PLP) cofactor. The second intermediate, with absorption features at approximately 417 and approximately 340 nm, is assigned as Cys aldimine and Cys ketimine forms in rapid equilibrium. In agreement with this assignment, a significant substrate alpha-deuterium equilibrium isotope effect ((2)H-EIE) favoring the aldimine form (417 nm) is observed in the second state produced in either wild-type CD0387 or the inactive C326A variant protein, which lacks the nucleophilic cysteine residue and is thus unable to proceed beyond this state unless "rescued" by a high concentration of an exogenous thiol. The third intermediate has an additional approximately 506 nm feature, characteristic of a quinonoid form, along with the features of the previous state. Its assignment as Ala aldimine, quinonoid, and ketimine forms in rapid equilibrium, which associates its formation with C-S bond cleavage and persulfide formation, is supported by its failure to develop in the C326A variant and the normal kinetic isotope effect ((2)H-KIE) on its formation, which is similar in magnitude to the (2)H-EIE disfavoring Cys-ketimine (from which the third state forms) in the second state. Decay of the Ala quinonoid absorption is tentatively attributed to a conformational change by the enzyme that disfavors this form in its equilibrium with Ala aldimine and Ala ketimine. Subsequent decay of the ketimine absorption ( approximately 340 nm) is attributed to release of Ala from the cofactor with an observed rate constant of 10 s(-1), the slowest step in the persulfide-forming half-reaction. The enzyme-persulfide.Ala complex dissociates rapidly with a K(d) of 98 mM. The final state with lambda(max) approximately 350 nm is assigned as a dead-end complex between the enzyme-persulfide and a second l-cysteine, which adds to the cofactor via its sulfhydryl group, possibly forming a cyclic thiazolidine species.
 ...
PMID:Kinetic analysis of cysteine desulfurase CD0387 from Synechocystis sp. PCC 6803: formation of the persulfide intermediate. 1988 76

NifS-like cysteine desulfurases are widespread enzymes involved in the mobilization of sulfur from cysteine. The genome of the filamentous diazotrophic cyanobacterium Anabaena PCC 7120 contains four open reading frames potentially encoding NifS-like proteins. One of them, alr2505, belongs to the pkn22 operon, which enables Anabaena to cope with oxidative stress. The Alr2505 protein was purified and found to share all the features characteristic of cysteine desufurases. This is the first NifS-like enzyme to be functionally characterized in this bacterium. On the basis of the transcriptional profiling of all nifS-like genes in Anabaena, it is concluded that alr2505 is the only cysteine desulfurase-encoding gene induced by oxidative stress. The function of Alr2505, which was termed OsiS, is discussed.
 ...
PMID:The alr2505 (osiS) gene from Anabaena sp. strain PCC7120 encodes a cysteine desulfurase induced by oxidative stress. 2068 87

Cysteine desulfurases, which supply sulfur for iron-sulfur cluster biogenesis, are broadly distributed in all phyla including cyanobacteria, the progenitors of plant chloroplasts. The SUF (sulfur utilization factor) system is responsible for Fe-S cluster biosynthesis under stress. The suf operon from cyanobacterium Anabaena PCC 7120 showed the presence of a cysteine desulfurase, sufS (alr2495), but not the accessory sulfur-accepting protein (SufE). However, an open reading frame (alr3513) encoding a SufE-like protein (termed AsaE, Anabaena sulfur acceptor E) was found at a location distinct from the suf operon. The purified SufS protein existed as a pyridoxal 5' phosphate (PLP)-containing dimer with a relatively low desulfurase activity. Interestingly, in the presence of the AsaE protein, the catalytic efficiency of this reaction increased 10-fold. In particular, for sulfur mobilization, the AsaE protein partnered only SufS and not other cysteine desulfurases from Anabaena. The SufS protein was found to physically interact with the AsaE protein, demonstrating that AsaE was indeed the missing partner of Anabaena SufS. The conserved cysteine of the SufS or the AsaE protein was essential for activity but not for their physical association. Curiously, overexpression of the SufS protein in Anabaena caused reduced formation of reactive oxygen species on exposure to hydrogen peroxide (H2O2), resulting in superior oxidative stress tolerance to the oxidizing agent when compared with the wild-type strain. Overall, the results highlight the functional interaction between the two proteins that mediate sulfur mobilization, in the cyanobacterial SUF pathway, and further reveal that overexpression of SufS can protect cyanobacteria from oxidative stress.
 ...
PMID:Molecular basis of function and the unusual antioxidant activity of a cyanobacterial cysteine desulfurase. 2859 83