57-60-3Relevant articles and documents
NON-IDENTITY OF CYSTINE LYASE WTH &β-CYSTATHIONASE IN TURNIP ROOTS
Mazelis, Mendel,Scott, Karen,Gallie, Daniel
, p. 991 - 996 (1982)
An active praparation of cystine lyase (EC 4.4.1-) was prepared from turnip roots and its substrate specificity examined.Only L-cysteine, cysteine-S-SO3, and the sulphoxides of L-djenkolic acid, S-methyl- and S-ethyl-L-cysteine were substrates.L-Cystathione, L-djenkolic acid, S-methyl- and S-ethyl-cysteines were not cleaved by this enzyme.The Km for L-cystine was 1.3 mM and L-cystathionine acted as an effective competitive inhibitor with a Ki of 0.7 mM.After dialysis against 10 mM potassium phosphate buffer pH 7.5, added pyridoxal phosphate was absolutely necessary for activity.In addition a marked stimulation was observed in the presence of ammonium sulphate.The products of the reaction were cysteine persulphide, pyruvate and presumably ammonia.The persulphide was easily demonstrated by cleavage with CN- to yield SCN- under conditions in which elemental sulphur was unreactive. Key Word Index- Brassica rapa,; Cruciferae; turnip root; cystine lyase; β-cystathionase; cystine degradation.
Converging conversion - using promiscuous biocatalysts for the cell-free synthesis of chemicals from heterogeneous biomass
Pick, André,Sieber, Volker,Sutiono, Samuel
supporting information, p. 3656 - 3663 (2021/06/06)
Production of chemicals from lignocellulosic biomass has been proposed as a suitable replacement to petrochemicals. However, one inherent challenge of biomass utilization is the heterogeneity of the substrate resulting in the presence of mixed sugars after hydrolysis. Fermentation of mixed sugars often leads to poor yield and generation of multiple by-products, thus complicating the subsequent downstream processing. System biocatalysis has thus been developed in recent years to address this challenge. In this work, several novel enzymes with broad substrate promiscuity were identified using a sequence-based discovery approach as suitable biocatalysts in a conversion ofd-xylose andl-arabinose, two major constituents of hemicellulose found in plant biomass. These promiscuous enzymes enabled simultaneous biotransformation ofd-xylose andl-arabinose to yield 1,4-butanediol (BDO) with a maximum production rate of 3 g L?1h?1and a yield of >95%. This model system was further adapted toward the production of α-ketoglutarate (2-KG) from the pentoses using O2as a cosubstrate for cofactor recycling reaching a maximum production rate of 4.2 g L?1h?1and a yield of 99%. To verify the potential applicability of our system, we attempted to scale up the BDO and 2-KG production fromd-xylose andl-arabinose. Simple optimization and reaction engineering allowed us to obtain BDO and 2-KG titers of 18 g L?1and 42 g L?1, with theoretical yields of >75% and >99%, respectively. One of the promiscuous enzymes identified together with auxiliary promiscuous enzymes was also suitable for stereoconvergent synthesis from a mixture ofd-glucose andd-galactose, predominant sugars found in food waste streams and microalgae biomass.
Conformational dynamics and allostery in pyruvate kinase
Donovan, Katherine A.,Zhu, Shaolong,Liuni, Peter,Peng, Fen,Kessans, Sarah A.,Wilson, Derek J.,Dobson, Renwick C.J.
, p. 9244 - 9256 (2016/05/19)
Pyruvate kinase catalyzes the final step in glycolysis and is allosterically regulated to control flux through the pathway. Two models are proposed to explain how Escherichia coli pyruvate kinase type 1 is allosterically regulated: the "domain rotation model" suggests that both the domains within the monomer and the monomers within the tetramer reorient with respect to one another; the "rigid body reorientation model" proposes only a reorientation of the monomers within the tetramer causing rigidification of the active site. To test these hypotheses and elucidate the conformational and dynamic changes that drive allostery, we performed time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange studies followed by mutagenic analysis to test the activation mechanism. Global exchange experiments, supported by thermostability studies, demonstrate that fructose 1, 6-bisphosphate binding to the allosteric domain causes a shift toward a globally more dynamic ensemble of conformations. Mapping deuterium exchange to peptides within the enzyme highlight site-specific regions with altered conformational dynamics, many of which increase in conformational flexibility. Based upon these and mutagenic studies, we propose an allosteric mechanism whereby the binding of fructose 1, 6-bisphosphate destabilizes an α-helix that bridges the allosteric and active site domains within the monomeric unit This destabilizes the βstrands within the (β/α)8-barrel domain and the linked active site loops that are responsible for substrate binding. Our data are consistent with the domain rotation model but inconsistent with the rigid body reorientation model given the increased flexibility at the interdomain interface, and we can for the first time explain how fructose 1, 6-bisphosphate affects the active site.
A role for glutamate-333 of Saccharomyces cerevisiae cystathionine γ-lyase as a determinant of specificity
Hopwood, Emily M.S.,Ahmed, Duale,Aitken, Susan M.
, p. 465 - 472 (2014/01/17)
Cystathionine γ-lyase (CGL) catalyzes the hydrolysis of l-cystathionine (l-Cth), producing l-cysteine (l-Cys), α-ketobutyrate and ammonia, in the second step of the reverse transsulfuration pathway, which converts l-homocysteine (l-Hcys) to l-Cys. Site-directed variants substituting residues E48 and E333 with alanine, aspartate and glutamine were characterized to probe the roles of these acidic residues, conserved in fungal and mammalian CGL sequences, in the active-site of CGL from Saccharomyces cerevisiae (yCGL). The pH optimum of variants containing the alanine or glutamine substitutions of E333 is increased by 0.4-1.2 pH units, likely due to repositioning of the cofactor and modification of the pKa of the pyridinium nitrogen. The pH profile of yCGL-E48A/E333A resembles that of Escherichia coli cystathionine β-lyase. The effect of substituting E48, E333 or both residues is the 1.3-3, 26-58 and 124-568-fold reduction, respectively, of the catalytic efficiency of l-Cth hydrolysis. The Kml-Cth of E333 substitution variants is increased ~ 17-fold, while Km l-OAS is within 2.5-fold of the wild-type enzyme, indicating that residue E333 interacts with the distal amine moiety of l-Cth, which is not present in the alternative substrate O-acetyl-l-serine. The catalytic efficiency of yCGL for α,γ-elimination of O-succinyl-l-homoserine (k cat/Kml-OSHS = 7 ± 2), which possesses a distal carboxylate, but lacks an amino group, is 300-fold lower than that of the physiological l-Cth substrate (kcat/Kml-Cth = 2100 ± 100) and 260-fold higher than that of l-Hcys (k cat/Kml-Hcys = 0.027 ± 0.005), which lacks both distal polar moieties. The results of this study suggest that the glutamate residue at position 333 is a determinant of specificity.
