The First Crystal Structure of l-Threonine Dehydrogenase

Ishikawa, Kazuhiko; Higashi, Noriko; Nakamura, Tsutomu; Matsuura, Takanori; Nakagawa, Atsushi

doi:10.1016/j.jmb.2006.11.060

Cited by 37 publications

(39 citation statements)

References 35 publications

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“…These values are in close agreement with kinetic constants reported for other TDH enzymes of eukaryotic origin (19,20). We conclude that the purified mouse TDH enzyme is capable of catalyzing the same chemical reaction as has been demonstrated for TDH orthologs from diverse metazoan and microbial species (19)(20)(21)(22).…”

Section: Resultssupporting

confidence: 67%

Targeted killing of a mammalian cell based upon its specialized metabolic state

Alexander

Wang

McKnight

2011

Proc. Natl. Acad. Sci. U.S.A.

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Mouse ES cells use a mitochondrial threonine dehydrogenase (TDH) enzyme to catabolize threonine into glycine and acetylCoA. Measurements of mRNA abundance have given evidence that ES cells express upwards of 1,000-fold higher levels of TDH mRNA than any of seven other mouse tissues tested. When cell culture medium is deprived of threonine, ES cells rapidly discontinue DNA synthesis, arrest cell division, and eventually die. Such studies led to the conclusion that mouse ES cells exist in a threonine-dependent metabolic state. Proceeding with the assumption that the active TDH enzyme should be essential for the growth and viability of mouse ES cells, we performed a drug screen in search of specific inhibitors of the purified TDH enzyme. Such efforts led to the discovery of a class of quinazolinecarboxamide (Qc) compounds that inhibit the ability of the TDH enzyme to catabolize threonine into glycine and acetyl-CoA. Administration of Qc inhibitors of TDH to mouse ES cells impeded cell growth and resulted in the induction of autophagy. By contrast, the same chemicals failed to affect the growth of HeLa cells at concentrations 300-fold higher than that required to kill mouse ES cells. It was likewise observed that the Qc class of TDH inhibitors failed to affect the growth or viability of ES cell-derived embryoid body cells known to have extinguished TDH expression. These studies demonstrate how it is possible to kill a specific mammalian cell type on the basis of its specialized metabolic state.specialized metabolism | threonine catabolism | embryonic stem cells T he diverse spectrum of life forms found in nature makes use of a variety of specialized metabolic pathways to support growth and reproduction. Over the past century, these often unanticipated specializations in metabolism have enabled scientists to pharmacologically poison specific organisms or cell types to the benefit of human health. One of the first examples of the utility of this strategy was the sulfonamide class of antibiotics. Discovered in the 1930s, sulfonamides act by inhibiting the bacterial enzyme dihydropteroate synthetase (DHPS) (1, 2). The DHPS enzyme is needed by bacteria to synthesize folic acid derivatives, which are required for the de novo synthesis of nucleotides. Mammals do not encode DHPS enzymes and instead obtain folates from their diet. For this reason sulfonamides are well tolerated by humans. A second example of an antibiotic that works by exploiting metabolic specialization is metronidazole. Metronidazole becomes toxic only after being reduced in the absence of oxygen; hence this chemical toxin selectively targets anaerobic microorganisms (3). Many drugs that block the growth of eukaryotic pathogens also work by attacking points of vulnerability built around specialized differences in metabolism. These include the azole, echinocandin, and allylamine classes of antifungal drugs (4-6), as well as the antiparasitics thiabendazole and diethylcarbazamine (7,8).Human cancer cells have also been targeted by virtue of their specialized...

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Section: Resultssupporting

confidence: 67%

Targeted killing of a mammalian cell based upon its specialized metabolic state

Alexander

Wang

McKnight

2011

Proc. Natl. Acad. Sci. U.S.A.

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“…Amino acid composition and its substitution patterns between mesophilic and hyperthermophilic proteins shed light on the understanding of common features of thermostability (56,63). T. guaymasensis ADH showed 77% identity to T. brockii ADH and The information refers to the recent crystal structures (11,19,20,27,30 (35). The uncharged polar residues Gln, Asn, and Ser decreased in T. guaymasensis ADH, in which the first two are prone to deamidation and known to be the most temperature sensitive (14,67).…”

Section: Discussionmentioning

confidence: 99%

“…Zinc-containing ADHs constitute a big protein family with various enzyme activities, including alcohol dehydrogenase, polyol dehydrogenase, and cinnamyl alcohol dehydrogenase activities (37). A large number of zinc-containing ADHs, including those from hyperthermophiles Thermococcus kodakaraensis, Pyrococcus horikoshii, Aeropyrum pernix, and Sulfolobus solfataricus, contain catalytic and structural zincs (5,11,20,25,30). The zinc-containing ADHs from the mesophile Clostridium beijerinckii and thermophiles Thermoanaerobacter brockii and Thermoanaerobacter ethanolicus contain only catalytic zinc (34).…”

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confidence: 99%

Characterization of a Zinc-Containing Alcohol Dehydrogenase with Stereoselectivity from the Hyperthermophilic Archaeon Thermococcus guaymasensis

Ying

2011

J Bacteriol

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An alcohol dehydrogenase (ADH) from hyperthermophilic archaeon Thermococcus guaymasensis was purified to homogeneity and was found to be a homotetramer with a subunit size of 40 ؎ 1 kDa. The gene encoding the enzyme was cloned and sequenced; this gene had 1,095 bp, corresponding to 365 amino acids, and showed high sequence homology to zinc-containing ADHs and L-threonine dehydrogenases with binding motifs of catalytic zinc and NADP ؉ . Metal analyses revealed that this NADP ؉ -dependent enzyme contained 0.9 ؎ 0.03 g-atoms of zinc per subunit. It was a primary-secondary ADH and exhibited a substrate preference for secondary alcohols and corresponding ketones. Particularly, the enzyme with unusual stereoselectivity catalyzed an anti-Prelog reduction of racemic (R/S)-acetoin to (2R,3R)-2,3-butanediol and meso-2,3-butanediol. The optimal pH values for the oxidation and formation of alcohols were 10.5 and 7.5, respectively. Besides being hyperthermostable, the enzyme activity increased as the temperature was elevated up to 95°C. The enzyme was active in the presence of methanol up to 40% (vol/vol) in the assay mixture. The reduction of ketones underwent high efficiency by coupling with excess isopropanol to regenerate NADPH. The kinetic parameters of the enzyme showed that the apparent K m values and catalytic efficiency for NADPH were 40 times lower and 5 times higher than those for NADP ؉ , respectively. The physiological roles of the enzyme were proposed to be in the formation of alcohols such as ethanol or acetoin concomitant to the NADPH oxidation.

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“…The functions of MDR family members are divergent (6), and this type of L-ThrDH is classified as a zinc-dependent MDR as well as an ADH. Several biochemical and structural studies have been performed on this type of L-ThrDH (3,(7)(8)(9)(10). The other type is classified within the extended short chain dehydrogenase/reductase (SDR) (4) and is similar to UDP-galactose 4-epimerase (GalE).…”

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confidence: 99%