3-hydroxyisobutyrate dehydrogenase-I from Pseudomonas denitrificans ATCC 13867 degrades 3-hydroxypropionic acid
3-hydroxyisobutyrate dehydrogenase-I from Pseudomonas denitrificans ATCC 13867 degrades 3-hydroxypropionic acid
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DOI：
10.1007/s12257-013-0487-x
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发表时间：
2014-02-01
影响因子：
3.2
通讯作者：
Park, Sunghoon
中科院分区：
文献类型：
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作者：
Lee, Philah;Raj, Subramanian Mohan;Park, Sunghoon
本研究考察了反硝化假单胞菌ATCC 13867的3-羟基异丁酸脱氢酶i （3HIBDHI）在3-羟丙酸（3-HP）生产过程中降解3-羟基丙酸（3-HP）的作用和生理相关性。克隆了P.反硝化菌ATCC 13867的3hibdh - 1基因，并在大肠杆菌BL21 （DE3）中表达。重组3hibdh - 1在Ni-NTA-HP柱上纯化，并对其选择的底物、辅助因子、金属、还原剂、最佳温度和ph进行了表征。重组3hibdh - 1对(S)-3-羟基异丁酸酯的催化常数(k （cat)/ k (m)）为604.1 +/- 71.1 mM/S，但对(R)-3-羟基异丁酸酯没有检测到活性。3hibdh - 1更倾向于NAD（+）而不是NADP（+）作为其催化活性的辅助因子。在37A℃、pH 9.0条件下，NAD（+）存在时，3-HP的k (cat)/ k (m)为15.40 +/- 1.43 mM/S。除了(S)-3-羟基异丁酸酯和3-HP外，3hibdh - 1还利用了l-丝氨酸、甲基-d、l-丝氨酸和甲基-(S)-(+)-3-羟基-2-甲基丙酸酯；另一方面，对这些衬底测定的k (cat)/ k (m)值小于5.0mM/S。乙二胺四乙酸、2-巯基乙醇、二硫苏糖醇和Mn2+显著提高了3HIBDHI的活性，而1.0 mM时Fe2+、Hg2+和Ag+的存在则完全抑制了其活性。本研究揭示了3hibdh - 1的特性及其在3-HP降解中的意义。
This study examined the role and physiological relevance of 3-hydroxyisobutyrate dehydrogenase-I (3HIBDHI) of Pseudomonas denitrificans ATCC 13867 in the degradation of 3-hydroxypropionic acid (3-HP) during 3-HP production. The gene encoding 3HIBDH-I of P. denitrificans ATCC 13867 was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant 3HIBDH-I was then purified on a Ni-NTA-HP column and characterized for its choice of substrates, cofactors, metals, reductants, and the optimal temperature and pH. The recombinant 3HIBDH-I showed a high catalytic constant (k (cat)/K (m)) of 604.1 +/- 71.1 mM/S on (S)-3-hydroxyisobutyrate, but no detectable activity on (R)-3-hydroxyisobutyrate. 3HIBDH-I preferred NAD(+) over NADP(+) as a cofactor for its catalytic activity. The k (cat)/K (m) determined for 3-HP was 15.40 +/- 1.43 mM/S in the presence of NAD(+) at 37A degrees C and pH 9.0. In addition to (S)-3-hydroxyisobutyrate and 3-HP, 3HIBDH-I utilized l-serine, methyl-d,l-serine, and methyl-(S)-(+)-3-hydroxy-2-methylpropionate; on the other hand, the k (cat)/K (m) values determined for these substrates were less than 5.0mM/S. Ethylenediaminetetraacetic acid, 2-mercaptoethanol, dithiothreitol and Mn2+ increased the activity of 3HIBDHI significantly, whereas the presence of Fe2+, Hg2+ and Ag+ in the reaction mixture at 1.0 mM completely inhibited its activity. This study revealed the characteristics of 3HIBDH-I and its significance in 3-HP degradation.