57-60-3Relevant academic research and scientific papers
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.
Purification and Characterization of Cystine Lyase a from Broccoli Inflorescence
Ukai, Koji,Sekiya, Jiro
, p. 1890 - 1895 (1997)
One of the three isoforms of an enzyme degrading L-cystine was purified to homogeneity from broccoli (Brassica oleracea var. italica) inflorescences, with use of a sensitive assay based on derivatization of a reaction product with monobromobimane. The reaction product with a thiol group was found to be thiocysteine from results of liquid chromatography-mass spectrometry and high-resolution mass spectrometry. Pyruvate was also a reaction product, formed in equimolar amounts. The purified enzyme catalyzed β-elimination of L-cystine to yield thiocysteine, pyruvate and possibly ammonia, so it was cystine lyase a. L-Cystine but not D-cystine was a substrate of the enzyme. S-Methyl L-cysteine sulfoxide and S-ethyl L-cysteine sulfoxide were substrates but were less suitable than L-cystine. L- and D-cysteine and also cystathionine were not substrates. The purified enzyme (Mr 186,000) was composed of four identical subunits (Mr 45,000) and was pyridoxal 5′-phosphate-dependent.
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.
Mechanistic Studies of the Streptomyces bingchenggensis Aldolase-Dehydratase: Implications for Substrate and Reaction Specificity in the Acetoacetate Decarboxylase-like Superfamily
Mydy, Lisa S.,Hoppe, Robert W.,Hagemann, Trevor M.,Schwabacher, Alan W.,Silvaggi, Nicholas R.
, p. 4136 - 4147 (2019/10/11)
The acetoacetate decarboxylase-like superfamily (ADCSF) is a little-explored group of enzymes that may contain new biocatalysts. The low level of sequence identity (～20%) between many ADCSF enzymes and the confirmed acetoacetate decarboxylases led us to investigate the degree of diversity in the reaction and substrate specificity of ADCSF enzymes. We have previously reported on Sbi00515, which belongs to Family V of the ADCSF and functions as an aldolase-dehydratase. Here, we more thoroughly characterize the substrate specificity of Sbi00515 and find that aromatic, unsaturated aldehydes yield lower KM and higher kcat values compared to those of other small electrophilic substrates in the condensation reaction. The roles of several active site residues were explored by site-directed mutagenesis and steady state kinetics. The lysine-glutamate catalytic dyad, conserved throughout the ADCSF, is required for catalysis. Tyrosine 252, which is unique to Sbi00515, is hypothesized to orient the incoming aldehyde in the condensation reaction. Transient state kinetics and an intermediate-bound crystal structure aid in completing a proposed mechanism for Sbi00515.
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.
Conversion of Anthranilate Synthase into Isochorismate Synthase: Implications for the Evolution of Chorismate-Utilizing Enzymes
Plach, Maximilian G.,L?ffler, Patrick,Merkl, Rainer,Sterner, Reinhard
supporting information, p. 11270 - 11274 (2016/07/06)
Chorismate-utilizing enzymes play a vital role in the biosynthesis of metabolites in plants as well as free-living and infectious microorganisms. Among these enzymes are the homologous primary metabolic anthranilate synthase (AS) and secondary metabolic i
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.
Structural Insights into the Recovery of Aldolase Activity in N-Acetylneuraminic Acid Lyase by Replacement of the Catalytically Active Lysine with γ-Thialysine by Using a Chemical Mutagenesis Strategy
Timms, Nicole,Windle, Claire L.,Polyakova, Anna,Ault, James R.,Trinh, Chi H.,Pearson, Arwen R.,Nelson, Adam,Berry, Alan
, p. 474 - 481 (2013/05/08)
Chemical modification has been used to introduce the unnatural amino acid γ-thialysine in place of the catalytically important Lys165 in the enzyme N-acetylneuraminic acid lyase (NAL). The Staphylococcus aureus nanA gene, encoding NAL, was cloned and expressed in E. coli. The protein, purified in high yield, has all the properties expected of a class I NAL. The S. aureus NAL which contains no natural cysteine residues was subjected to site-directed mutagenesis to introduce a cysteine in place of Lys165 in the enzyme active site. Subsequently chemical mutagenesis completely converted the cysteine into γ-thialysine through dehydroalanine (Dha) as demonstrated by ESI-MS. Initial kinetic characterisation showed that the protein containing γ-thialysine regained 17% of the wild-type activity. To understand the reason for this lower activity, we solved X-ray crystal structures of the wild-type S. aureus NAL, both in the absence of, and in complex with, pyruvate. We also report the structures of the K165C variant, and the K165-γ-thialysine enzyme in the presence, or absence, of pyruvate. These structures reveal that γ-thialysine in NAL is an excellent structural mimic of lysine. Measurement of the pH-activity profile of the thialysine modified enzyme revealed that its pH optimum is shifted from 7.4 to 6.8. At its optimum pH, the thialysine-containing enzyme showed almost 30% of the activity of the wild-type enzyme at its pH optimum. The lowered activity and altered pH profile of the unnatural amino acid-containing enzyme can be rationalised by imbalances of the ionisation states of residues within the active site when the pKa of the residue at position 165 is perturbed by replacement with γ-thialysine. The results reveal the utility of chemical mutagenesis for the modification of enzyme active sites and the exquisite sensitivity of catalysis to the local structural and electrostatic environment in NAL.
Entropic and enthalpic components of catalysis in the mutase and lyase activities of pseudomonas aeruginosa PchB
Luo, Qianyi,Meneely, Kathleen M.,Lamb, Audrey L.
experimental part, p. 7229 - 7233 (2011/06/23)
The isochorismate-pyruvate lyase from Pseudomonas aeruginosa (PchB) catalyzes two pericyclic reactions, demonstrating the eponymous activity and also chorismate mutase activity. The thermodynamic parameters for these enzyme-catalyzed activities, as well a
PH dependence of catalysis by pseudomonas aeruginosa isochorismate - Pyruvate lyase: Implications for transition state stabilization and the role of lysine 42
Olucha, Jose,Ouellette, Andrew N.,Luo, Qianyi,Lamb, Audrey L.
experimental part, p. 7198 - 7207 (2012/07/13)
An isochorismate - pyruvate lyase with adventitious chorismate mutase activity from Pseudomonas aerugionsa (PchB) achieves catalysis of both pericyclic reactions in part by the stabilization of reactive conformations and in part by electrostatic transitio
