4-Methylpentanoate

• 分子结构分类: 有机化合物 - 脂质和类脂质分子 - 脂肪酰基
• 英文名称: 4-Methylpentanoate
• 化学式: C6H11O2-
• 分子量: 115.15
• InChiKey: FGKJLKRYENPLQH-UHFFFAOYSA-M

计算性质

• 辛醇/水分配系数(LogP)：
  2.1
• 重原子数：
  8
• 可旋转键数：
  2
• 环数：
  0.0
• sp3杂化的碳原子比例：
  0.83
• 拓扑面积：
  40.1
• 氢给体数：
  0
• 氢受体数：
  2

反应信息

• 作为反应物：
  描述：

  4-Methylpentanoate 、 adenosine 5'-triphosphate 、 coenzyme A 生成 4-methylpentanoyl-CoA(4-) 、 adenosine 5'-monophosphate 、 pyrophosphoric acid
  参考文献：
  名称：

  Characterization of the Formation of Branched Short-Chain Fatty Acid:CoAs for Bitter Acid Biosynthesis in Hop Glandular Trichomes

  摘要：

  Two cytosol CoA ligases and one mitochondrial thioesterase were biochemically characterized from hop glandular trichomes. The present data provide an insight into the understanding of hop bitter acid biosynthesis and tools for the microbial bioengineering of bitter acids.Bitter acids, known for their use as beer flavoring and for their diverse biological activities, are predominantly formed in hop (Humulus lupulus) glandular trichomes. Branched short-chain acyl-CoAs (e.g. isobutyryl-CoA, isovaleryl-CoA and 2-methylbutyryl-CoA), derived from the degradation of branched-chain amino acids (BCAAs), are essential building blocks for the biosynthesis of bitter acids in hops. However, little is known regarding what components are needed to produce and maintain the pool of branched short-chain acyl-CoAs in hop trichomes. Here, we present several lines of evidence that both CoA ligases and thioesterases are likely involved in bitter acid biosynthesis. Recombinant HlCCL2 (carboxyl CoA ligase) protein had high specific activity for isovaleric acid as a substrate (K-cat/K-m 4100 s(1) M-1), whereas recombinant HlCCL4 specifically utilized isobutyric acid (K-cat/K-m 1800 s(1) M-1) and 2-methylbutyric acid (K-cat/K-m 6900 s(1) M-1) as substrates. Both HlCCLs, like hop valerophenone synthase (HlVPS), were expressed strongly in glandular trichomes and localized to the cytoplasm. Co-expression of HlCCL2 and HlCCL4 with HlVPS in yeast led to significant production of acylphloroglucinols (the direct precursors for bitter acid biosynthesis), which further confirmed the biochemical function of these two HlCCLs in vivo. Functional identification of a thioesterase that catalyzed the reverse reaction of CCLs in mitochondria, together with the comprehensive analysis of genes involved BCAA catabolism, supported the idea that cytosolic CoA ligases are required for linking BCAA degradation and bitter acid biosynthesis in glandular trichomes. The evolution and other possible physiological roles of branched short-chain fatty acid:CoA ligases in planta are also discussed.

  DOI：

  10.1093/mp/sst004
• 作为产物：
  描述：

  (R)-2-Hydroxy-4-methyl-pentanoic acid anion 、 4-methylpentanoyl-CoA(4-) 生成 (R)-2-hydroxy-4-methylpentanoyl-CoA(4-) 、 4-Methylpentanoate
  参考文献：
  名称：

  Characterization of (R)-2-Hydroxyisocaproate Dehydrogenase and a Family III Coenzyme A Transferase Involved in Reduction ofl-Leucine to Isocaproate byClostridium difficile

  摘要：

  摘要人体肠道中存在一种严格厌氧的致病性艰难梭状芽孢杆菌，它能通过发酵亮氨酸而繁殖。通过发酵，氨基酸被氧化成异戊酸加二氧化碳，又被还原成异己酸。在这一途径的还原分支中，(R)-2-羟基异己氨酰辅酶 A（CoA）脱水成(E)-2-异己氨酰辅酶 A（CoA）可能是通过自由基中间体催化的。脱水酶需要通过依赖 ATP 的单电子转移激活（J. Kim、D. Darley 和 W. Buckel，FEBS J. 272:550-561, 2005）。在脱水之前，脱氢酶和 CoA 转移酶应该参与形成 (R)-2-羟基异己基-CoA。从艰难梭菌基因组中推导出的ldhA和hadA的氨基酸序列分别与乳酸脱氢酶和III族CoA转移酶具有很高的相似性。克隆了编码脱氢酶和 CoA 转移酶的两个推定基因，并在大肠杆菌中进行了过表达；纯化了重组的 Strep 标记 II 融合蛋白，并对其进行了鉴定。单体 LdhA（36.5 kDa）的底物特异性表明，2-氧代异己酸酯（Km= 68 μM，kcat= 31 s-1）和 NADH 是原生底物。在逆反应中，该酶接受(R)-而不接受(S)-2-羟基异己酸酯，因此被命名为(R)-2-羟基异己酸酯脱氢酶。HadA 显示了以(R)-2-羟基异己基-CoA 为供体、异己酸或(E)-2-异己烯酸为受体的 CoA 转移酶活性。通过定点突变，发现保守的 D171 是一个重要的催化残基，可能参与了硫酯底物酰基混合酸酐的形成。然而，无论是羟胺还是硼氢化钠（这两种物质都是 CoA 转移酶的失活剂）都没有改变这个残基。脱氢酶和 CoA 转移酶完全符合将亮氨酸还原为异己酸酯的拟议途径。

  DOI：

  10.1128/aem.00772-06

文献信息

• Functional Screening and <i>In Vitro</i> Analysis Reveal Thioesterases with Enhanced Substrate Specificity Profiles That Improve Short-Chain Fatty Acid Production in Escherichia coli
  作者：Matthew D. McMahon、Kristala L. J. Prather

  DOI：10.1128/aem.03303-13

  日期：2014.2

  Short-chain fatty acid (SCFA) biosynthesis is pertinent to production of biofuels, industrial compounds, and pharmaceuticals from renewable resources. To expand on Escherichia coli SCFA products, we previously implemented a coenzyme A (CoA)-dependent pathway that condenses acetyl-CoA to a diverse group of short-chain fatty acyl-CoAs. To increase product titers and reduce premature pathway termination products, we conducted in vivo and in vitro analyses to understand and improve the specificity of the acyl-CoA thioesterase enzyme, which releases fatty acids from CoA. A total of 62 putative bacterial thioesterases, including 23 from the cow rumen microbiome, were inserted into a pathway that condenses acetyl-CoA to an acyl-CoA molecule derived from exogenously provided propionic or isobutyric acid. Functional screening revealed thioesterases that increase production of saturated (valerate), unsaturated ( trans -2-pentenoate), and branched (4-methylvalerate) SCFAs compared to overexpression of E. coli thioesterase tesB or native expression of endogenous thioesterases. To determine if altered thioesterase acyl-CoA substrate specificity caused the increase in product titers, six of the most promising enzymes were analyzed in vitro . Biochemical assays revealed that the most productive thioesterases rely on promiscuous activity but have greater specificity for product-associated acyl-CoAs than for precursor acyl-CoAs. In this study, we introduce novel thioesterases with improved specificity for saturated, branched, and unsaturated short-chain acyl-CoAs, thereby expanding the diversity of potential fatty acid products while increasing titers of current products. The growing uncertainty associated with protein database annotations denotes this study as a model for isolating functional biochemical pathway enzymes in situations where experimental evidence of enzyme function is absent.

  摘要 短链脂肪酸（SCFA）的生物合成与利用可再生资源生产生物燃料、工业化合物和药物有关。为了扩展 大肠杆菌 SCFA 产品，我们先前采用了一种依赖辅酶 A（CoA）的途径，该途径可将乙酰-CoA 缩合为多种短链脂肪酰-CoAs。为了提高产物滴度并减少过早的途径终止产物，我们进行了 体内 和 体外 分析，以了解并改进从 CoA 中释放脂肪酸的酰基-CoA 硫代酯酶的特异性。总共有 62 种推测的细菌硫酯酶（包括 23 种来自牛瘤胃微生物组的细菌硫酯酶）被插入到将乙酰-CoA 缩合成酰基-CoA 分子的途径中，该酰基-CoA 分子来自外源提供的丙酸或异丁酸。功能筛选结果显示，硫酯酶可增加饱和（戊酸）、不饱和（反式）和乙酰-CoA 分子的产生。 反式 -2-戊烯酸）和支链（4-甲基戊酸）SCFAs 的产生。 大肠杆菌 硫酯酶 tesB 或内源性硫酯酶的原生表达相比。为了确定硫酯酶酰基-CoA 底物特异性的改变是否会导致产物滴度的增加，对六种最有前景的酶进行了分析 体外 .生化测定显示，产量最高的硫酯酶依赖于杂合活性，但对产物相关酰基-CoAs 的特异性比对前体酰基-CoAs 的特异性更高。在本研究中，我们引入了新型硫酯酶，它对饱和、支链和不饱和短链酰基-CoAs 的特异性更强，从而扩大了潜在脂肪酸产物的多样性，同时提高了现有产物的滴度。由于蛋白质数据库注释的不确定性越来越大，因此本研究可作为在缺乏酶功能实验证据的情况下分离功能性生化途径酶的一种模式。