Characterization of two isotypes of L-threonine dehydratase from Entamoeba histolytica
Characterization of two isotypes of L-threonine dehydratase from Entamoeba histolytica
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DOI：
10.1016/j.molbiopara.2009.11.004
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发表时间：
2010-04-01
影响因子：
1.5
通讯作者：
Nozaki, Tomoyoshi
中科院分区：
文献类型：
--
作者：
Husain, Afzal;Jeelani, Ghulam;Nozaki, Tomoyoshi
肠道原虫溶组织内阿米巴的基因组序列表明，氨基酸分解代谢在能量代谢中起着重要作用。在本研究中，我们描述了溶组织肠杆菌分解代谢的L-苏氨酸和L-丝氨酸脱水酶(TD)的动力学和调节性质。TD催化L-苏氨酸和L-丝氨酸依赖于磷酸吡哆醛的脱水脱氨反应，生成氨和酮酸(分别为2-氧丁酸和丙酮酸)。溶组乳杆菌有两个TD亚型(EhTd1-2)，具有38%的同源性，计算的分子质量分别为45.0和46.5 kDa，等电点分别为6.68和5.88。只有EhTd1显示了L-苏氨酸和L-丝氨酸的脱水脱氨活性，而EhTD2没有这些活性，因为底物中涉及的氨基酸残基和辅因子结合不保守。L-苏氨酸的K(CAT)/K-m值是L-丝氨酸的3倍。L-半胱氨酸对EhTd1呈竞争性抑制，L-丝氨酸和L-苏氨酸的K-I值分别为1.1mM和2.2mM。EhTd1对AMP或CMP的变构激活不敏感。EhTD1的三个主要替换可能归因于这种不敏感。20 mM 2-氧丁酸、丙酮酸和乙醛对EhTD1的抑制作用约为50%，但这种抑制作用不被AMP逆转。综上所述，这些数据表明，EhTD1具有不同于其他生物的独特调节特性，可能通过降解氨基酸在溶组织性肠杆菌的能量代谢中发挥重要作用。(C)2009爱思唯尔B.V.保留所有权利。
The genome sequence of the enteric protozoan parasite Entamoeba histolytica suggests that amino acid catabolism plays an important role in energy metabolism. In the present study, we described kinetic and regulatory properties of catabolic L-threonine and L-serine dehydratase (TD) from E. histolytica. TD catalyses the pyridoxal phosphate-dependent dehydrative deamination Of L-threonine and L-serine to ammonia and keto acids (2-oxobutyrate and pyruvate, respectively). E. histolytica possesses two TD isotypes (EhTD1-2) showing 38% Mutual identity, a calculated molecular mass of 45.0 or 46.5 kDa, and an isoelectric point of 6.68 or 5.88, respectively. Only EhTD1 showed L-threonine and L-serine dehydrative deaminating activities whereas EhTD2, in which the amino acid residues involved in the substrate and cofactor binding were not conserved, was devoid of these activities. The k(cat)/K-m value of EhTD1 was >3 fold higher for L-threonine than L-serine. EhTD1 was inhibited by L-Cysteine in a competitive manner with the K-i values of 1.1 mM and 2.2 mM for L-serine and L-threonine, respectively. EhTD1 was insensitive to the allosteric activation by AMP or CMP. Three major substitutions of EhTD1 likely attribute to the insensitivity. EhTD1 was also inhibited about 50% by 20 mM 2-oxobutyrate, pyruvate, and glyoxylate; the inhibition was not, however, reversed by AMP. Together, these data showed that EhTD1 possesses unique regulatory properties distinct from other organisms and may play an important role in energy metabolism via amino acid degradation in E. histolytica. (C) 2009 Elsevier B.V. All rights reserved.