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首页> 外文学位 >Alternative pathway for provision of acyl CoA precursors for fatty acid biosynthesis: Purification and kinetic characterization of phosphotransbutyrylase and butyrate kinase from Listeria monocytogenes
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Alternative pathway for provision of acyl CoA precursors for fatty acid biosynthesis: Purification and kinetic characterization of phosphotransbutyrylase and butyrate kinase from Listeria monocytogenes

机译：提供用于脂肪酸生物合成的酰基CoA前体的替代途径：单核细胞增生性李斯特菌中磷酸转丁酰酶和丁酸激酶的纯化和动力学表征

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摘要

Listeria monocytogenes is a foodborne pathogen that causes listeriosis, a disease characterized by gastroenteritis, meningitis, spontaneous miscarriages and high mortality rate among infected individuals. L. monocytogenes is a major concern in the food industry, due to its ability to grow at refrigeration temperatures and the zero tolerance policy of the FDA, resulting in expensive food recalls. Growth at low temperatures is aided in part by the high membrane content of branched-chain fatty acids (BCFAs) which imparts greater fluidity to the membrane. Mutants of L. monocytogenes impaired in BCFA biosynthesis display diminished growth at normal and low temperatures, exhibit lower tolerance to acidity and alkalinity, and demonstrate lower virulence. Addition of 2-methylbutyrate, the source of the membrane BCFA anteiso C15:0, restores the membrane BCFA content and virulence factor expression. Supplementation with unnatural branched-chain carboxylic acids (BCCAs) such as 2- ethylbutyrate and 2-methylpentanoate results in the incorporation of novel BCFAs in the listerial membrane. Incorporation of supplemented carboxylic acids is evidence of their entry into the fatty acid biosynthesis pathway and thus a Bkd-independent pathway which catalyzes the conversion of these compounds into their activated CoA derivatives must exist in L. monocytogenes. We hypothesize that Ptb and Buk, the products of the first two genes of the bkd operon, are involved in the sequential conversion of the supplemented BCCAs into their acyl CoA derivatives, which then presumably enter the fatty acid biosynthesis pathway for elongation. Ptb catalyzes the reversible conversion of acyl CoAs into acyl phosphates and Buk catalyzes the reversible phosphorylation of carboxylic acids. Ptb and Buk were heterologously expressed in E. coli, purified by affinity chromatography and utilized for analysis of their kinetic properties to determine their role in the activation of such carboxylic acids. Ptb and Buk both demonstrate broad substrate specificity and do not use acetate or hexanoate efficiently as substrates. Ptb and Buk exhibit a strong preference for substrates which have a chain length of C3-C5 thus indicating that they are not involved either in acetate metabolism or in the activation of long chain fatty acids. Ptb shows a strong preference for branched-chain substrates while Buk appears to demonstrate preference for BCCAs only with respect to C3 and C4 substrates. Both Ptb and Buk from L. monocytogenes demonstrate significant activity with unnatural BCCAs such as 2-ethylbutyrate and 2-methylpentanoate. Additionally, Buk exhibits substantial phosphorylation activity at low temperatures and appears to prefer BCCAs thus demonstrating a switch in substrate specificities at low temperatures. Ptb catalysis involves the formation of a ternary complex with acyl CoA and phosphate before release of the products. Similarly, Buk also forms a ternary complex with carboxylic acid and ATP before catalysis and release of products. Our work here indicates that Ptb and Buk likely act in a sequential manner in the activation and subsequent assimilation of exogenous straight chain carboxylic acids (SCCAs) and BCCAs.
机译：单核细胞增生李斯特菌是引起李斯特菌病的食源性病原体，李斯特菌病的特征是胃肠炎，脑膜炎，自然流产和高感染率。单核细胞增生李斯特氏菌是食品工业中的一个主要问题，因为它在冷藏温度下具有生长的能力以及FDA的零容忍政策，导致召回昂贵的食品。低温下的生长部分是由于支链脂肪酸（BCFA）的膜含量高，从而使膜具有更大的流动性。 BCFA生物合成中受损的单核细胞增生李斯特氏菌突变体在常温和低温下显示出生长减慢，对酸度和碱度的耐受性较低，并显示出较低的毒力。膜BCFA反异源C15：0的来源2-甲基丁酸酯的添加可恢复膜BCFA含量和毒力因子表达。补充非天然支链羧酸（BCCA）（例如2-乙基丁酸酯和2-甲基戊酸酯）会导致新的BCFA进入李斯特菌膜中。补充羧酸的结合是它们进入脂肪酸生物合成途径的证据，因此在单核细胞增生李斯特氏菌中必须存在不依赖Bkd的途径，该途径催化这些化合物向其活化的CoA衍生物的转化。我们假设bkd操纵子的前两个基因的产物Ptb和Buk参与了将补充的BCCA顺序转换为它们的酰基CoA衍生物的过程，然后可能进入脂肪酸生物合成途径进行延伸。 Ptb催化酰基CoAs的可逆转化为酰基磷酸酯，而Buk催化羧酸的可逆磷酸化。 Ptb和Buk在大肠杆菌中异源表达，通过亲和层析纯化，并用于分析其动力学性质，以确定它们在此类羧酸活化中的作用。 Ptb和Buk都显示出广泛的底物特异性，并且没有有效地使用乙酸盐或己酸作为底物。 Ptb和Buk对链长为C3-C5的底物表现出强烈的偏爱，因此表明它们既不参与乙酸酯代谢，也不参与长链脂肪酸的活化。 Ptb对支链底物表现出强烈的偏好，而Buk似乎仅对C3和C4底物表现出对BCCA的偏好。单核细胞增生李斯特氏菌的Ptb和Buk均表现出对非天然BCCA（例如2-乙基丁酸和2-甲基戊酸）的显着活性。另外，Buk在低温下表现出显着的磷酸化活性，并且似乎更喜欢BCCA，因此证明了低温下底物特异性的变化。 Ptb催化涉及在释放产物之前与酰基CoA和磷酸盐形成三元络合物。同样，Buk在催化和释放产物之前也与羧酸和ATP形成三元络合物。我们在这里的工作表明，Ptb和Buk在外源直链羧酸（SCCA）和BCCA的活化和随后的同化中可能以顺序的方式起作用。

著录项

  • 作者

    Sirobhushanam, Sirisha.;

  • 作者单位

    Illinois State University.;

  • 授予单位 Illinois State University.;
  • 学科 Molecular biology.;Microbiology.;Biochemistry.
  • 学位 Ph.D.
  • 年度 2016
  • 页码 86 p.
  • 总页数 86
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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