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A Priming Cassette Generates Hydroxylated Acyl Starter Units in Mupirocin and Thiomarinol Biosynthesis.
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2020-02-06 , DOI: 10.1021/acschembio.9b00969 Paul D Walker 1, 2 , Matthew T Rowe 1 , Ashley J Winter 1 , Angus N M Weir 1 , Nahida Akter 1 , Luoyi Wang 1 , Paul R Race 3 , Christopher Williams 1 , Zhongshu Song 1 , Thomas J Simpson 1 , Christine L Willis 1 , Matthew P Crump 1
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2020-02-06 , DOI: 10.1021/acschembio.9b00969 Paul D Walker 1, 2 , Matthew T Rowe 1 , Ashley J Winter 1 , Angus N M Weir 1 , Nahida Akter 1 , Luoyi Wang 1 , Paul R Race 3 , Christopher Williams 1 , Zhongshu Song 1 , Thomas J Simpson 1 , Christine L Willis 1 , Matthew P Crump 1
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Mupirocin, a commercially available antibiotic produced by Pseudomonas fluorescens NCIMB 10586, and thiomarinol, isolated from the marine bacterium Pseudoalteromonas sp. SANK 73390, both consist of a polyketide-derived monic acid homologue esterified with either 9-hydroxynonanoic acid (mupirocin, 9HN) or 8-hydroxyoctanoic acid (thiomarinol, 8HO). The mechanisms of formation of these deceptively simple 9HN and 8HO fatty acid moieties in mup and tml, respectively, remain unresolved. To define starter unit generation, the purified mupirocin proteins MupQ, MupS, and MacpD and their thiomarinol equivalents (TmlQ, TmlS and TacpD) have been expressed and shown to convert malonyl coenzyme A (CoA) and succinyl CoA to 3-hydroxypropionoyl (3-HP) or 4-hydroxybutyryl (4-HB) fatty acid starter units, respectively, via the MupQ/TmlQ catalyzed generation of an unusual bis-CoA/acyl carrier protein (ACP) thioester, followed by MupS/TmlS catalyzed reduction. Mix and match experiments show MupQ/TmlQ to be highly selective for the correct CoA. MacpD/TacpD were interchangeable but alternate trans-acting ACPs from the mupirocin pathway (MacpA/TacpA) or a heterologous ACP (BatA) were nonfunctional. MupS and TmlS selectivity was more varied, and these reductases differed in their substrate and ACP selectivity. The solution structure of MacpD determined by NMR revealed a C-terminal extension with partial helical character that has been shown to be important for maintaining high titers of mupirocin. We generated a truncated MacpD construct, MacpD_T, which lacks this C-terminal extension but retains an ability to generate 3-HP with MupS and MupQ, suggesting further downstream roles in protein-protein interactions for this region of the ACP.
中文翻译：
入门盒在莫匹罗星和硫代马林醇的生物合成中生成羟基化的酰基起始剂单元。
从海洋细菌假单胞菌属物种中分离出的莫匹罗星（一种由荧光假单胞菌NCIMB 10586生产的可商购抗生素）和硫代水醇。SANK 73390均由用9-羟基壬酸（莫匹罗星，9HN）或8-羟基辛酸（硫代马来醇，8HO）酯化的聚酮化合物衍生的一元酸同系物组成。尚未在mup和tml中分别形成这些看似简单的9HN和8HO脂肪酸部分的机制尚不清楚。为了定义起始单元的生成，已经表达了纯化的莫匹罗星蛋白MupQ，MupS和MacpD及其硫代马林醇等效物（TmlQ，TmlS和TacpD），并显示将丙二酰辅酶A（CoA）和琥珀酰CoA转化为3-羟基丙酰（3- HP）或4-羟基丁酰基（4-HB）脂肪酸起始剂单元，通过MupQ / TmlQ催化生成不寻常的双CoA /酰基载体蛋白（ACP）硫酯，然后通过MupS / TmlS催化还原。混合和匹配实验表明，MupQ / TmlQ对正确的CoA具有高度的选择性。MacpD / TacpD是可互换的，但来自莫匹罗星途径（MacpA / TacpA）或异源ACP（BatA）的交替反式ACP没有功能。MupS和TmlS的选择性变化更大，这些还原酶的底物和ACP选择性也不同。通过NMR确定的MacpD的溶液结构显示具有部分螺旋特征的C端延伸，这对于维持高滴度的莫匹罗星很重要。我们生成了一个截断的MacpD结构MacpD_T，该结构缺少C端扩展，但保留了使用MupS和MupQ生成3-HP的能力，
更新日期：2020-02-06
中文翻译：
入门盒在莫匹罗星和硫代马林醇的生物合成中生成羟基化的酰基起始剂单元。
从海洋细菌假单胞菌属物种中分离出的莫匹罗星（一种由荧光假单胞菌NCIMB 10586生产的可商购抗生素）和硫代水醇。SANK 73390均由用9-羟基壬酸（莫匹罗星，9HN）或8-羟基辛酸（硫代马来醇，8HO）酯化的聚酮化合物衍生的一元酸同系物组成。尚未在mup和tml中分别形成这些看似简单的9HN和8HO脂肪酸部分的机制尚不清楚。为了定义起始单元的生成，已经表达了纯化的莫匹罗星蛋白MupQ，MupS和MacpD及其硫代马林醇等效物（TmlQ，TmlS和TacpD），并显示将丙二酰辅酶A（CoA）和琥珀酰CoA转化为3-羟基丙酰（3- HP）或4-羟基丁酰基（4-HB）脂肪酸起始剂单元，通过MupQ / TmlQ催化生成不寻常的双CoA /酰基载体蛋白（ACP）硫酯，然后通过MupS / TmlS催化还原。混合和匹配实验表明，MupQ / TmlQ对正确的CoA具有高度的选择性。MacpD / TacpD是可互换的，但来自莫匹罗星途径（MacpA / TacpA）或异源ACP（BatA）的交替反式ACP没有功能。MupS和TmlS的选择性变化更大，这些还原酶的底物和ACP选择性也不同。通过NMR确定的MacpD的溶液结构显示具有部分螺旋特征的C端延伸，这对于维持高滴度的莫匹罗星很重要。我们生成了一个截断的MacpD结构MacpD_T，该结构缺少C端扩展，但保留了使用MupS和MupQ生成3-HP的能力，
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