Utilization of multiple substrates by butyrate kinase from Listeria monocytogenes.
Utilization of multiple substrates by butyrate kinase from Listeria monocytogenes.
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单核细胞增生李斯特菌丁酸激酶利用多种底物。
DOI：
10.1016/j.bbalip.2016.12.001
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Gatto,Craig
中科院分区：
文献类型：
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作者：
Sirobhushanam,Sirisha;Galva,Charitha;Saunders,LaurenP;Sen,Suranjana;Jayaswal,Radheshyam;Wilkinson,BrianJ;Gatto,Craig
单核细胞增生李斯特菌，致病菌的李斯特菌，可以建立了危险的水平，在冷藏食品可能导致昂贵的产品召回。细菌在低温下生长的一个重要方面是其在较低生长温度下增加其膜的支链脂肪酸anteiso C15：0含量的能力，这赋予更大的膜流动性。支链α-酮脱氢酶（bkd）复合物中的突变体缺乏支链脂肪酸（BCFA），但这些可以通过喂食C4和C5支链羧酸（BCCA）来恢复。这表明存在用于脂肪酸生物合成的酰基CoA前体的替代途径。我们推测交替途径由bkd复合物编码的丁酸激酶（布克）和磷酸转丁酰酶（ptb）组成，它们通过羧酸代谢的顺序作用产生酰基CoA产物。我们使用11种不同的直链和BCCA底物在酰基磷酸形成方向上测定了重组His标记的布克的稳态动力学。布克表现出最高的催化效率与戊酸作为底物。用乙酸盐（C2）和己酸盐（C6）作为底物观察到的低产物形成表明，布克不参与乙酸盐代谢或长链羧酸活化。我们还能够表明，布克催化发生通过三元复合物中间体。此外，布克在低温下表现出对BCCAs的强烈偏好。这些结果表明，布克可能参与了外源羧酸的活化和同化膜脂肪酸的生物合成。
Listeria monocytogenes, the causative agent of listeriosis, can build up to dangerous levels in refrigerated foods potentially leading to expensive product recalls. An important aspect of the bacterium's growth at low temperatures is its ability to increase the branched-chain fatty acid anteiso C15:0 content of its membrane at lower growth temperatures, which imparts greater membrane fluidity. Mutants in the branched-chain α-keto dehydrogenase (bkd) complex are deficient in branched-chain fatty acids (BCFAs,) but these can be restored by feeding C4 and C5 branched-chain carboxylic acids (BCCAs). This suggests the presence of an alternate pathway for production of acyl CoA precursors for fatty acid biosynthesis. We hypothesize that the alternate pathway is composed of butyrate kinase (buk) and phosphotransbutyrylase (ptb) encoded in thebkdcomplex which produce acyl CoA products by their sequential action through the metabolism of carboxylic acids. We determined the steady state kinetics of recombinant His-tagged Buk using 11 different straight-chain and BCCA substrates in the acyl phosphate forming direction. Buk demonstrated highest catalytic efficiency with pentanoate as the substrate. Low product formation observed with acetate (C2) and hexanoate (C6) as the substrates indicates that Buk is not involved in either acetate metabolism or long chain carboxylic acid activation. We were also able to show that Buk catalysis occurs through a ternary complex intermediate. Additionally, Buk demonstrates a strong preference for BCCAs at low temperatures. These results indicate that Buk may be involved in the activation and assimilation of exogenous carboxylic acids for membrane fatty acid biosynthesis.