Pre-steady-state Kinetic and Structural Analysis of Interaction of Methionine γ-Lyase from Citrobacter freundii with Inhibitors

Kuznetsov, Nikita А.; Faleev, N. G.; Кузнецова, А. А.; Morozova, Elena A.; Revtovich, Svetlana V.; Anufrieva, Natalya V.; Nikulin, Alexei; Fedorova, Olga S.; Demidkina, Tatyana V.

doi:10.1074/jbc.m114.586511

Cited by 19 publications

(14 citation statements)

References 41 publications

(49 reference statements)

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“…This finding was also confirmed for the inactivation of γ -aminobutyric (GABA) aminotransferase by L-cycloserine (LCS). 7 Structures of the DCS- and LCS-inhibited forms of dialkylglycine decarboxylase 8 and of the LCS-inhibited forms of methionine γ -lyase 9 have also been reported.…”

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confidence: 98%

Mechanism-Based Inhibition of the Mycobacterium tuberculosis Branched-Chain Aminotransferase by d- and l-Cycloserine

et al. 2017

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The branched-chain aminotransferase is a pyridoxal 5′-phosphate (PLP)-dependent enzyme responsible for the final step in the biosynthesis of all three branched-chain amino acids, L-leucine, L-isoleucine, and L-valine, in bacteria. We have investigated the mechanism of inactivation of the branched-chain aminotransferase from Mycobacterium tuberculosis (MtIlvE) by D- and L-cycloserine. D-Cycloserine is currently used only in the treatment of multidrug–drug-resistant tuberculosis. Our results show a time-and concentration-dependent inactivation of MtIlvE by both isomers, with L-cycloserine being a 40-fold better inhibitor of the enzyme. Minimum inhibitory concentration (MIC) studies revealed that L-cycloserine is a 10-fold better inhibitor of Mycobacterium tuberculosis growth than D-cycloserine. In addition, we have crystallized the MtIlvE-D-cycloserine inhibited enzyme, determining the structure to 1.7 Å. The structure of the covalent D-cycloserine-PMP adduct bound to MtIlvE reveals that the D-cycloserine ring is planar and aromatic, as previously observed for other enzyme systems. Mass spectrometry reveals that both the D-cycloserine- and L-cycloserine-PMP complexes have the same mass, and are likely to be the same aromatized, isoxazole product. However, the kinetics of formation of the MtIlvE D-cycloserine-PMP and MtIlvE L-cycloserine-PMP adducts are quite different. While the kinetics of the formation of the MtIlvE D-cycloserine-PMP complex can be fit to a single exponential, the formation of the MtIlvE L-cycloserine-PMP complex occurs in two steps. We propose a chemical mechanism for the inactivation of D- and L-cycloserine which suggests a stereochemically determined structural role for the differing kinetics of inactivation. These results demonstrate that the mechanism of action of D-cycloserine’s activity against M. tuberculosis may be more complicated than previously thought and that D-cycloserine may compromise the in vivo activity of multiple PLP-dependent enzymes, including MtIlvE.

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confidence: 98%

Mechanism-Based Inhibition of the Mycobacterium tuberculosis Branched-Chain Aminotransferase by d- and l-Cycloserine

et al. 2017

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“…46 L-Cycloserine (LCS) is a synthetic compound, which inhibits γ-aminobutyric acid aminotransferase, dialkylglycine decarboxylase, and methionine γ-lyase. 43,47,48 More recently, Franco et al elucidated the mechanism of inhibition of MtIlvE, a branched-chain aminotransferase and PLP-dependent enzyme involved in the last step of the biosynthetic pathway of BCAAs, by DCS and LCS. 49 Because DCS and LCS are known inhibitors of PLPdependent enzymes, both compounds were tested on MsIlvA.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Biochemical Characterization of the Mycobacterium smegmatis Threonine Deaminase

2018

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The biosynthesis of branched-chain amino acids or BCAAs (l-isoleucine, l-leucine, and l-valine) is essential in eubacteria, but mammals are branched-chain amino acid auxotrophs, making the enzymes in the pathway excellent targets for antibacterial drug development. The biosynthesis of l-isoleucine, l-leucine, and l-valine is very efficient, requiring only eight enzymes. Threonine dehydratase (TD), a pyridoxal 5'-phosphate (PLP)-dependent enzyme encoded by the ilvA gene, is the enzyme responsible for the conversion of l-threonine (l-Thr) to α-ketobutyrate, ammonia, and water, which is the first step in the biosynthesis of l-isoleucine. We have cloned, expressed, and biochemically characterized the reaction catalyzed by Mycobacterium smegmatis TD (abbreviated as MsIlvA) using steady-state kinetics and kinetic isotope effects. We show here that in addition to l-threonine, l-allo-threonine and l-serine are also used as substrates by TD, and all exhibit sigmoidal, non-Michaelis-Menten kinetics. Curiously, β-chloro-l-alanine was also a substrate rather than an inhibitor as expected. The enzymatic activity of TD is sensitive to the presence of allosteric regulators, including the activator l-valine or the end product feedback inhibitor of the BCAA pathway in which TD is involved, l-isoleucine. Primary deuterium kinetic isotopes are small, suggesting Cα proton abstraction is only partially rate-limiting. Solvent kinetic isotopes were significantly larger, indicating that a proton transfer occurring during the reaction is also partially rate-limiting. Finally, we demonstrate that l-cycloserine, a general inhibitor of PLP-dependent enzymes, is an excellent inhibitor of threonine deaminase.

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“…For purification of STR3, a His-tag encoding fragment was included before the stop codon (TTA). After the PCR product was cloned into pYES2, the resulting plasmid, pYES2-STR3, was transformed into S. cerevisiae strain INVSc1 as described 24 . Transformants were selected in SC medium lacking uracil (SC-U) and further confirmed by colony PCR with universal primers of F5 and R3.…”

Section: Methodsmentioning

confidence: 99%

“…More importantly, methioninase transfected into cancer cells evinced either moderate cell death or severe aggregation 18 . However, these methioninases are of bacterial origin, and usually associated with high immunogenicity, low substrate specificity, and hazardous effects on the kidney and liver 17 22 23 24 . The proposed methioninase in fungi needs to be functionally defined for its potential to overcome these defects.…”

mentioning

confidence: 99%

Clonostachys rosea demethiolase STR3 controls the conversion of methionine into methanethiol

Jia

Zhang

Sun

et al. 2016

Sci Rep

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Eukaryote-derived methioninase, catalyzing the one-step degradation of methionine (Met) to methanethiol (MTL), has received much attention for its low immunogenic potential and use as a therapeutic agent against Met-dependent tumors. Although biological and chemical degradation pathways for Met-MTL conversion are proposed, the concrete molecular mechanism for Met-MTL conversion in eukaryotes is still unclear. Previous studies demonstrated that α-keto-methylthiobutyric acid (KMBA), the intermediate for Met-MTL conversion, was located extracellularly and the demethiolase STR3 possessed no activities towards Met, which rule out the possibility of intracellular Met-MTL conversion pathway inside eukaryotes. We report here that degradation of Met resulted in intracellular accumulation of KMBA in Clonostachys rosea. Addition of Met to culture media led to the production of MTL and downregulation of STR3, while incubation of Met with surrogate substrate α-ketoglutaric acid enhanced the synthesis of MTL and triggered the upregulation of STR3. Subsequent biochemical analysis with recombinant STR3 showed that STR3 directly converted both Met and its transamination product KMBA to MTL. These results indicated that STR3 as rate-limiting enzyme degrades Met and KMBA into MTL. Our findings suggest STR3 is a potential target for therapeutic agents against Met-dependent tumors and aging.

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Pre-steady-state Kinetic and Structural Analysis of Interaction of Methionine γ-Lyase from Citrobacter freundii with Inhibitors

Cited by 19 publications

References 41 publications

Mechanism-Based Inhibition of the Mycobacterium tuberculosis Branched-Chain Aminotransferase by d- and l-Cycloserine

Mechanism-Based Inhibition of the Mycobacterium tuberculosis Branched-Chain Aminotransferase by d- and l-Cycloserine

Biochemical Characterization of the Mycobacterium smegmatis Threonine Deaminase

Clonostachys rosea demethiolase STR3 controls the conversion of methionine into methanethiol

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