26062-47-5Relevant articles and documents
Stereochemical Course of the Reaction Catalyzed by RimO, a Radical SAM Methylthiotransferase
Landgraf, Bradley J.,Booker, Squire J.
, p. 2889 - 2892 (2016)
RimO is a member of the growing radical S-adenosylmethionine (SAM) superfamily of enzymes, which use a reduced [4Fe-4S] cluster to effect reductive cleavage of the 5′ C-S bond of SAM to form a 5′-deoxyadenosyl 5′-radical (5′-dA? intermediate. RimO uses this potent oxidant to catalyze the attachment of a methylthio group (-SCH3) to C3 of aspartate 89 of protein S12, one of 21 proteins that compose the 30S subunit of the bacterial ribosome. However, the exact mechanism by which this transformation takes place has remained elusive. Herein, we describe the stereochemical course of the RimO reaction. Using peptide mimics of the S12 protein bearing deuterium at the 3 pro-R or 3 pro-S positions of the target aspartyl residue, we show that RimO from Bacteroides thetaiotaomicron (Bt) catalyzes abstraction of the pro-S hydrogen atom, as evidenced by the transfer of deuterium into 5′-deoxyadenosine (5′-dAH). The observed kinetic isotope effect on H atom versus D atom abstraction is ～1.9, suggesting that this step is at least partially rate determining. We also demonstrate that Bt RimO can utilize the flavodoxin/flavodoxin oxidoreductase/NADPH reducing system from Escherichia coli as a source of requisite electrons. Use of this in vivo reducing system decreases, but does not eliminate, formation of 5′-dAH in excess of methylthiolated product.
Mechanism of stereospecific conversion of DL-5-substituted hydantoins to the corresponding L-amino acids by Pseudomonas sp. strain NS671
Ishikawa, Takahiro,Watabe, Ken,Mukohara, Yukuo,Nakamura, Hiroaki
, p. 185 - 187 (1997)
The mechanism of stereospecific conversion of DBL-5-substituted hydantoins to the corresponding L-amino acids by Pseudomonas sp. strain NS671 was studied. The results indicated that the hydantoinase catalyzed the hydrolysis reaction of both D- and L-5-(2-methylthioethyl)hydantoin, and that the hydrolysis of the L-enantiomer proceeded preferentially compared with that of the D-enantiomer. On the basis of these findings, the mechanism was speculated to be as follows: DBL-5-substituted hydantoins are converted exclusively to the L-forms of the corresponding N-carbamyl-amino acids by the hydantoinase in combination with hydantoin racemase. The N-carbamyl-L-amino acids are then converted to L-amino acids by N-carbamyl-L-amino acid amidohydrolase.
Increasing the synthesis/hydrolysis ratio of aminoacylase 1 by site-directed mutagenesis
Wardenga, Rainer,Lindner, Holger A.,Hollmann, Frank,Thum, Oliver,Bornscheuer, Uwe
, p. 102 - 109 (2010)
Aminoacylase-1 from pig kidney (pAcy1) catalyzes the highly stereoselective acylation of amino acids, a useful conversion for the preparation of optically pure N-acyl-l-amino acids. The kinetic of this thermodynamically controlled conversion is determined by maximal velocities for synthesis (VmS) and hydrolysis (VmH) of the N-acyl-l-amino acid. To investigate which parameter affects maximal velocities, we focused on?the proton acceptor potential of the catalytic base, E146, and studied the influence of the active site architecture on its contribution to the pKa of residue E146. The modeled structure of pAcy1 identified residue D346 as having the strongest impact on the electrostatic features of the catalytic base. Substitutions of D346 generally decreased enzymatic activities but also altered both the pH-dependency of hydrolytic activity and the VmS/VmH ratio of pAcy1. A reduced theoretical pKa value and a lowered experimental pH optimum of hydrolytic rates for the D346A mutant were associated with a 9-fold increase in VmS/VmH. This?supports the importance of electrostatic contributions of D346 to the acid-base properties of E146 and demonstrates for the first time the possibility of engineering the VmS/VmH ratio of pAcy1.
Functional characterization of methionine sulfoxide reductase A from Trypanosoma spp.
Arias, Diego G.,Cabeza, Matías S.,Erben, Esteban D.,Carranza, Pedro G.,Lujan, Hugo D.,I?ón, María T. Téllez,Iglesias, Alberto A.,Guerrero, Sergio A.
, p. 37 - 46 (2011)
Methionine is an amino acid susceptible to being oxidized to methionine sulfoxide (MetSO). The reduction of MetSO to methionine is catalyzed by methionine sulfoxide reductase (MSR), an enzyme present in almost all organisms. In trypanosomatids, the study of antioxidant systems has been mainly focused on the involvement of trypanothione, a specific redox component in these organisms. However, no information is available concerning their mechanisms for repairing oxidized proteins, which would be relevant for the survival of these pathogens in the various stages of their life cycle. We report the molecular cloning of three genes encoding a putative A-type MSR in trypanosomatids. The genes were expressed in Escherichia coli, and the corresponding recombinant proteins were purified and functionally characterized. The enzymes were specific for L-Met(S)SO reduction, using Trypanosoma cruzi tryparedoxin I as the reducing substrate. Each enzyme migrated in electrophoresis with a particular profile reflecting the differences they exhibit in superficial charge. The in vivo presence of the enzymes was evidenced by immunological detection in replicative stages of T. cruzi and Trypanosoma brucei. The results support the occurrence of a metabolic pathway in Trypanosoma spp. involved in the critical function of repairing oxidized macromolecules.
Structure of Candida albicans methionine synthase determined by employing surface residue mutagenesis
Ubhi, Devinder,Kavanagh, Kathryn L.,Monzingo, Arthur F.,Robertus, Jon D.
, p. 19 - 26 (2011)
Fungal methionine synthase, Met6p, transfers a methyl group from 5-methyl-tetrahydrofolate to homocysteine to generate methionine. The enzyme is essential to fungal growth and is a potential anti-fungal drug design target. We have characterized the enzyme from the pathogen Candida albicans but were unable to crystallize it in native form. We converted Lys103, Lys104, and Glu107 all to Tyr (Met6pY), Thr (Met6pT) and Ala (Met6pA). All variants showed wild-type kinetic activity and formed useful crystals, each with unique crystal packing. In each case the mutated residues participated in beneficial crystal contacts. We have solved the three structures at 2.0-2.8 resolution and analyzed crystal packing, active-site residues, and similarity to other known methionine synthase structures. C. albicans Met6p has a two domain structure with each of the domains having a (βα)8-barrel fold. The barrels are arranged face-to-face and the active site is located in a cleft between the two domains. Met6p utilizes a zinc ion for catalysis that is bound in the C-terminal domain and ligated by four conserved residues: His657, Cys659, Glu679 and Cys739.
Light-Driven Kinetic Resolution of α-Functionalized Carboxylic Acids Enabled by an Engineered Fatty Acid Photodecarboxylase
Xu, Jian,Hu, Yujing,Fan, Jiajie,Arkin, Mamatjan,Li, Danyang,Peng, Yongzhen,Xu, Weihua,Lin, Xianfu,Wu, Qi
, p. 8474 - 8478 (2019)
Chiral α-functionalized carboxylic acids are valuable precursors for a variety of medicines and natural products. Herein, we described an engineered fatty acid photodecarboxylase (CvFAP)-catalyzed kinetic resolution of α-amino acids and α-hydroxy acids, which provides the unreacted R-configured substrates with high yields and excellent stereoselectivity (ee up to 99 %). This efficient light-driven process requires neither NADPH recycling nor prior preparation of esters, which were required in previous biocatalytic approaches. The structure-guided engineering strategy is based on the scanning of large amino acids at hotspots to narrow the substrate binding tunnel. To the best of our knowledge, this is the first example of asymmetric catalysis by an engineered CvFAP.
Asymmetric carboxylation in the synthesis of L-methionine: A new tool for 11C chemistry
Jeanjean, Fabien,Perol, Nathalie,Gore, Jacques,Fournet, Guy
, p. 7547 - 7550 (1997)
L-methionine is obtained in good yield and high e.e. by using the carboxylation of an enantiopure α-lithio oxazolidinone prepared by tin-lithium exchange. The entire process from α-stannyl oxazolidinone takes 35-40mn, time which is compatible with the use of 11CO2 in radioactive chemistry directed to PET imaging.
Increasing the storage and oxidation stabilities of N-acyl-d-amino acid amidohydrolase by site-directed mutagenesis of critical methionine residues
Peng, I-Chen,Lo, Kai-Yin,Hsu, Chun-Hua,Lee, Chia-Yin
, p. 1785 - 1790 (2012)
The recombinant N-acyl-d-amino acid amidohydrolase (N-d-AAase) of Variovorax paradoxus Iso1 was unstable during protein purification and storage at 4 °C. Since the methionine oxidation might be the artificial factor leading to the inactivation of N-d-AAase, eight potential oxidation sensitive methionine residues of the enzyme were individually substituted with leucine utilizing site-directed mutagenesis. Among them, five mutants, M39L, M56L, M221L, M254L, and M352L remained at least 70% of wild-type specific activity. The enzyme kinetic parameters of M221L revealed a 44% decrease in Km, and finally reflected a 2.4-fold increase in kcat/Km. Moreover, its half-life at 4 °C increased up to 6-fold longer than that of the wild-type. Structural analysis of each methionine substitution was carried out based on the crystal structure of N-d-AAase from Alcaligenes faecalis DA1. Met221 spatial closeness to the zinc-assistant catalytic center is highly potential as the primary site for oxidative inactivation. We conclude that the replacement of methionine M221 with leucine in N-d-AAase successfully enhances the oxidative resistance, half-life, and enzyme activity. This finding provides a promising basis for the engineering the stability and activity of N-d-AAase.
Analyses of methionine sulfoxide reductase activities towards free and peptidyl methionine sulfoxides
Kwak, Geun-Hee,Kim, Hwa-Young,Hwang, Kwang Yeon
, p. 1 - 5,5 (2012)
There have been insufficient kinetic data that enable a direct comparison between free and peptide methionine sulfoxide reductase activities of either MsrB or MsrA. In this study, we determined the kinetic parameters of mammalian and yeast MsrBs and MsrAs for the reduction of both free methionine sulfoxide (Met-O) and peptidyl Met-O under the same assay conditions. Catalytic efficiency of mammalian and yeast MsrBs towards free Met-O was >2000-fold lower than that of yeast fRMsr, which is specific for free Met-R-O. The ratio of free to peptide Msr activity in MsrBs was 1:20-40. In contrast, mammalian and yeast MsrAs reduced free Met-O much more efficiently than MsrBs. Their kcat values were 40-500-fold greater than those of the corresponding MsrBs. The ratio of free to peptide Msr activity was 1:0.8 in yeast MsrA, indicating that this enzyme can reduce free Met-O as efficiently as peptidyl Met-O. In addition, we analyzed the in vivo free Msr activities of MsrBs and MsrAs in yeast cells using a growth complementation assay. Mammalian and yeast MsrBs, as well as the corresponding MsrAs, had apparent in vivo free Msr activities. The in vivo free Msr activities of MsrBs and MsrAs agreed with their in vitro activities.
Methionine sulfoxide reductases preferentially reduce unfolded oxidized proteins and protect cells from oxidative protein unfolding
Tarrago, Lionel,Kaya, Alaattin,Weerapana, Eranthie,Marino, Stefano M.,Gladyshev, Vadim N.
, p. 24448 - 24459 (2012)
Reduction of methionine sulfoxide (MetO) residues in proteins is catalyzed by methionine sulfoxide reductases A (MSRA) and B (MSRB), which act in a stereospecific manner. Catalytic properties of these enzymes were previously established mostly using low molecular weight MetO-containing compounds, whereas little is known about the catalysis ofMetOreduction in proteins, the physiological substrates ofMSRAand MSRB. In this work we exploited an NADPH-dependent thioredoxin system and determined the kinetic parameters of yeastMSRAandMSRBusing three different MetO-containing proteins. Both enzymes showed Michaelis-Menten kinetics with the Km lower for protein than for small MetO-containing substrates. MSRA reduced both oxidized proteins and low molecular weight MetO-containing compounds with similar catalytic efficiencies, whereasMSRBwas specialized for the reduction of MetO in proteins. Using oxidized glutathione S-transferase as a model substrate, we showed that both MSR types were more efficient in reducing MetO in unfolded than in folded proteins and that their activities increased with the unfolding state. Biochemical quantificationandidentificationofMetOreducedinthesubstrates by mass spectrometry revealed that the increased activity was due to better access to oxidized MetO in unfolded proteins; it also showed that MSRA was intrinsically more active with unfolded proteins regardless of MetO availability. Moreover, MSRs most efficiently protected cells from oxidative stress that was accompanied by protein unfolding. Overall, this study indicates that MSRs serve a critical function in the folding process by repairing oxidatively damaged nascent polypeptides and unfolded proteins.
