The High-resolution Structure of LeuB (Rv2995c) from Mycobacterium tuberculosis

Singh, Rajesh; Kefala, Georgia; Janowski, Robert; Mueller-Dieckmann, Christoph; Kries, Jens-Peter von; Weiss, M.S.

doi:10.1016/j.jmb.2004.11.059

Cited by 25 publications

(42 citation statements)

References 35 publications

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“…128 Sequences of IPMDH from E. coli and S. typhimurium are 94.5% identical, while the sequences of these enzymes are 51% identical with that of Thermus thermophilus IPMDH. 127 The overall topology of all three proteins is however very similar.…”

Section: Leub Isopropylmalate Dehydrogenasementioning

confidence: 93%

Bacterial Branched-Chain Amino Acid Biosynthesis: Structures, Mechanisms, and Drugability

2017

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The eight enzymes responsible for the biosynthesis of the three branched-chain amino acids (l-isoleucine, l-leucine, and l-valine) were identified decades ago using classical genetic approaches based on amino acid auxotrophy. This review will highlight the recent progress in the determination of the three-dimensional structures of these enzymes, their chemical mechanisms, and insights into their suitability as targets for the development of antibacterial agents. Given the enormous rise in bacterial drug resistance to every major class of antibacterial compound, there is a clear and present need for the identification of new antibacterial compounds with nonoverlapping targets to currently used antibacterials that target cell wall, protein, mRNA, and DNA synthesis.

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Section: Leub Isopropylmalate Dehydrogenasementioning

confidence: 93%

Bacterial Branched-Chain Amino Acid Biosynthesis: Structures, Mechanisms, and Drugability

2017

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“…1A). Multiple structures of IPMDH from bacteria indicate that the structural features around the active site are flexible and that active site dynamics likely plays a potential role in substrate recognition and catalysis (27). Moreover, the effect of the longer side-chain on the kinetics of the various glucosinolate biosynthesis pathway enzymes (i.e.…”

Section: -Isopropylmalate 3-(2-methylthio)ethylmalatementioning

confidence: 99%

Structural and Functional Evolution of Isopropylmalate Dehydrogenases in the Leucine and Glucosinolate Pathways of Arabidopsis thaliana

Galant

Pang

et al. 2011

Journal of Biological Chemistry

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The methionine chain-elongation pathway is required for aliphatic glucosinolate biosynthesis in plants and evolved from leucine biosynthesis. In Arabidopsis thaliana, three 3-isopropylmalate dehydrogenases (AtIPMDHs) play key roles in methionine chain-elongation for the synthesis of aliphatic glucosinolates (e.g. AtIPMDH1) and leucine (e.g. AtIPMDH2 and AtIPMDH3). Here we elucidate the molecular basis underlying the metabolic specialization of these enzymes. The 2.25 Å resolution crystal structure of AtIPMDH2 was solved to provide the first detailed molecular architecture of a plant IPMDH. Modeling of 3-isopropylmalate binding in the AtIPMDH2 active site and sequence comparisons of prokaryotic and eukaryotic IPMDH suggest that substitution of one active site residue may lead to altered substrate specificity and metabolic function. Sitedirected mutagenesis of Phe-137 to a leucine in AtIPMDH1 (AtIPMDH1-F137L) reduced activity toward 3-(2-methylthio)-ethylmalate by 200-fold, but enhanced catalytic efficiency with 3-isopropylmalate to levels observed with AtIPMDH2 and AtIPMDH3. Conversely, the AtIPMDH2-L134F and AtIPMDH3-L133F mutants enhanced catalytic efficiency with 3-(2-methylthio)ethylmalate ϳ100-fold and reduced activity for 3-isopropylmalate. Furthermore, the altered in vivo glucosinolate profile of an Arabidopsis ipmdh1 T-DNA knock-out mutant could be restored to wild-type levels by constructs expressing AtIPMDH1, AtIPMDH2-L134F, or AtIPMDH3-L133F, but not by AtIPMDH1-F137L. These results indicate that a single amino acid substitution results in functional divergence of IPMDH in planta to affect substrate specificity and contributes to the evolution of specialized glucosinolate biosynthesis from the ancestral leucine pathway.

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“…[7]. It is known that 3IPMDH has an absolute requirement of divalent cations for its activity and that monovalent cations significantly enhance the enzyme activity [4,6,7,8]. Amongst divalent cations tested we observed that Mn ++ is the most efficacious, followed by Zn ++ and Mg ++ , whereas Ca ++ is unable to sustain any enzymatic activity ( Table 2).…”

Section: Kinetic Analysis and Properties Of Recombinant Hi-3ipmdhmentioning

confidence: 74%

“…Protein expression was induced by adding isopropyl--D-thiogalactoside to a final concentration of 0.3 mM and by prolonging bacterial growth for 3 hours at 30 °C. Bacteria were then harvested by centrifugation (8,000 g for 15 min at 4 °C) and resuspended in 50 ml of lysis solution consisting of 50 mM phosphate buffer, pH 7.0 and protease inhibitors cocktail (from Sigma; contains: 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), bestatin, pepstatin A, E-64, and phosphoramidon). After ultrasonication on ice, the bacterial lysate was clarified by centrifugation (15,000 g for 45 min at 4 °C) and ammonium sulphate was added to the resulting supernatant (40% saturation).…”

Section: Expression and Purification Of Recombinant H Influenzae 3ipmdhmentioning

confidence: 99%

“…1) [6]. Both steps are absolutely dependent on the presence of a divalent cation and monovalent cations were reported to significantly enhance catalysis [4,6,7,8]. In yeast, the genes encoding leucine biosynthetic enzymes are located on different chromosomes [3,9] whereas in bacteria the four genes constitute a single operon, leuABCD, that is transcriptionally regulated by leucine [3,10,11].…”

Section: Introductionmentioning

confidence: 99%

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Expression, Purification and Characterisation of Haemophilus influenzae 3-Isopropylmalate Dehydrogenase (LeuB)

Martignon¹,

Rossi²,

Rizzi

2007

PPL

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3-isopropylmalate dehydrogenase (3IPMDH) is the third enzyme in leucine biosynthesis and a promising target for the development of broad-spectrum antibacterial agents. We report here the expression, purification and biochemical characterisation of Haemophilus influenzae 3-isopropylmalate dehydrogenase. The observed enzyme inhibition by the reaction product NADH could represent a regulatory mechanism for 3IPMDH.

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The High-resolution Structure of LeuB (Rv2995c) from Mycobacterium tuberculosis

Cited by 25 publications

References 35 publications

Bacterial Branched-Chain Amino Acid Biosynthesis: Structures, Mechanisms, and Drugability

Bacterial Branched-Chain Amino Acid Biosynthesis: Structures, Mechanisms, and Drugability

Structural and Functional Evolution of Isopropylmalate Dehydrogenases in the Leucine and Glucosinolate Pathways of Arabidopsis thaliana

Expression, Purification and Characterisation of Haemophilus influenzae 3-Isopropylmalate Dehydrogenase (LeuB)

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