A price to pay for relaxed substrate specificity: a comparative kinetic analysis of the class II lanthipeptide synthetases ProcM and HalM2.
A price to pay for relaxed substrate specificity: a comparative kinetic analysis of the class II lanthipeptide synthetases ProcM and HalM2.
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DOI：
10.1021/ja5089452
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发表时间：
2014-12-17
影响因子：
15
通讯作者：
van der Donk, Wilfred A.
中科院分区：
文献类型：
--
作者：
Thibodeaux, Christopher J.;Ha, Taekjip;van der Donk, Wilfred A.
羊毛硫肽是一类核糖体合成和后修饰的肽天然产物（RIPPs），其通常具有多个分子内硫醚键。对于II类羊毛硫肽，这些交联通过单一酶（LanM）安装在多步反应途径中。LanM的多功能性质和其遗传编码的肽底物（LanAs）的可操纵性使得LanM/拉娜系统成为新抗菌化合物工程化的有希望的靶标。在这里，我们报告了一个半定量的质谱为基础的测定动力学表征的LanM催化反应的发展。该测定用于进行两种LanM酶（HalM 2和ProcM）的比较动力学分析，这两种酶表现出显著不同的底物选择性。动力学数据的数值模拟被用来开发多步HalM 2和ProcM催化反应的模型。这些模型说明HalM 2和ProcM对于它们催化的各种反应具有显著不同的催化效率。HalM 2负责单一化合物（lantibiotic haloduracin的Halβ亚基）的生物合成，催化反应的催化效率高于ProcM，ProcM在普罗氯红生物合成过程中修饰29种不同的ProcA前体肽。特别地，硫醚环形成的速率在ProcM中急剧降低，可能是因为这种酶负责在其普罗氯欣产物中安装各种羊毛硫肽环结构。因此，ProcM似乎为其放松的底物特异性付出了动力学代价。此外，我们的动力学模型表明，构象采样的LanM/拉娜米氏复合物可以发挥重要作用的动力学拉娜成熟。
Lanthipeptides are a class of ribosomally synthesized and posttranslationally modified peptide natural products (RiPPs) that typically harbor multiple intramolecular thioether linkages. For class II lanthipeptides, these cross-links are installed in a multistep reaction pathway by a single enzyme (LanM). The multifunctional nature of LanMs and the manipulability of their genetically encoded peptide substrates (LanAs) make LanM/LanA systems promising targets for the engineering of new antibacterial compounds. Here, we report the development of a semiquantitative mass spectrometry-based assay for kinetic characterization of LanM-catalyzed reactions. The assay was used to conduct a comparative kinetic analysis of two LanM enzymes (HalM2 and ProcM) that exhibit drastically different substrate selectivity. Numerical simulation of the kinetic data was used to develop models for the multistep HalM2- and ProcM-catalyzed reactions. These models illustrate that HalM2 and ProcM have markedly different catalytic efficiencies for the various reactions they catalyze. HalM2, which is responsible for the biosynthesis of a single compound (the Halβ subunit of the lantibiotic haloduracin), catalyzes reactions with higher catalytic efficiency than ProcM, which modifies 29 different ProcA precursor peptides during prochlorosin biosynthesis. In particular, the rates of thioether ring formation are drastically reduced in ProcM, likely because this enzyme is charged with installing a variety of lanthipeptide ring architectures in its prochlorosin products. Thus, ProcM appears to pay a kinetic price for its relaxed substrate specificity. In addition, our kinetic models suggest that conformational sampling of the LanM/LanA Michaelis complex could play an important role in the kinetics of LanA maturation.
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影响因子：
15
作者：
Levengood, Matthew R.;Knerr, Patrick J.;Oman, Trent J.;van der Donk, Wilfred A.
通讯作者：
van der Donk, Wilfred A.
影响因子：
15
作者：
Levengood, Matthew R.;Patton, Gregory C.;van der Donk, Wilfred A.
通讯作者：
van der Donk, Wilfred A.
DOI：
10.1073/pnas.0913677107
发表时间：
2010-06-08
影响因子：
11.1
作者：
Li, Bo;Sher, Daniel;van der Donk, Wilfred A.
通讯作者：
van der Donk, Wilfred A.
影响因子：
4.4
作者：
Bierbaum, G;Szekat, C;Sahl, HG
通讯作者：
Sahl, HG
影响因子：
2.9
作者：
Johnson, Kenneth A.;Simpson, Zachary B.;Blom, Thomas
通讯作者：
Blom, Thomas