Insights into the mechanism of the antibiotic-synthesizing enzyme MoeO5 from crystal structures of different complexes.
Insights into the mechanism of the antibiotic-synthesizing enzyme MoeO5 from crystal structures of different complexes.
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DOI：
10.1002/anie.201108002
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发表时间：
2012-04-23
影响因子：
16.6
通讯作者：
Guo, Rey-Ting
中科院分区：
文献类型：
--
作者：
Ren, Feifei;Ko, Tzu-Ping;Feng, Xinxin;Huang, Chun-Hsiang;Chan, Hsiu-Chien;Hu, Yumei;Wang, Ke;Ma, Yanhe;Liang, Po-Huang;Wang, Andrew H. -J.;Oldfield, Eric;Guo, Rey-Ting
磷酸糖脂类抗生素莫诺霉素通过抑制肽聚糖糖基转移酶直接阻断细菌细胞壁的生物合成MoeO5酶由加纳链霉菌（Streptomyces ghanaensis）中moe基因簇1编码，催化莫诺霉素生产的第一步，其中法尼酯焦磷酸（FPP）的c15 -碳氢部分转移到3-磷酸甘油酸（3PG）的2-羟基上，形成醚键（方案1）该反应类似于香叶酰甘油磷酸合成酶（GGGPS）催化合成古菌型磷脂的反应然而，与GGGPS酶不同的是，它的产物保留了异戊烯基链的全反式构型，而MoeO5酶在转移的法尼基的C2= C3双键处发生了反式到顺式异构化蛋白质GGGPS和细菌同源物PcrB的晶体结构显示了三磷酸异构酶（TIM）桶状折叠，这在以前的戊烯基转移酶中没有观察到。[3,5] GGGPS和PcrB序列的氨基酸同源性为35%，而MoeO5与这两种酶的氨基酸同源性仅为10%（见支持信息中的图S1）。本文报道了MoeO5与产物2-(Z， E)-法尼基-3-磷酸甘油酸酯（FPG）、底物类似物法尼基硫代磷酸酯（FsPP）、镁（Mg2+）和焦磷酸盐（PPi）结合的x射线晶体结构；结合其他生化和生物信息学结果，这些结构揭示了这种不寻常酶的可能作用机制。使用GGGPS和PcrB作为搜索模型来确定MoeO5结构的分子替代方法并不成功，因此可能反映了蛋白质序列的显着差异。由于MoeO5不含Cys残基，为了利用多重同构置换（MIR）解决结构问题，我们制备了突变体H97C，用于高效制备汞基MIR衍生物（支持信息中表S1）。其他结构采用分子置换法求解（支持信息和表1中图S2，详见支持信息）。MoeO5结晶为同型二聚体（支持信息中的图3和表2）。二聚体界面在每个单体上埋埋了超过10%表面积的12002，主要包括螺旋a4和a5中的疏水残基。Phe140-Pro141的顺式肽在分子二联体上与一个Mg2+结合（支持信息中的图S3）。这些螺旋也介导GGGPS和PcrB中的二聚化，但在MoeO5中，其中一个TIM桶旋转了1808（支持信息中的图S4）。关于蛋白质结构以及结合配体的更详细的描述可以在辅助信息中找到。尽管MoeO5与GGGPS和PcrB单体的Ca根均方差（rmsd）约为2.0（见支持信息中的图S5），但在链b3和链b4之间具有连接环（表示为l3）的相似蛋白折叠清楚地将MoeO5置于这类新的TIM-barrel戊烯基转移酶中。
The phosphoglycolipid antibiotic moenomycin directly blocks bacterial cell-wall biosynthesis by inhibiting peptidoglycan glycosyltransferases.[1] The enzyme MoeO5, encoded by the moe gene cluster 1 in Streptomyces ghanaensis, catalyzes the initial step of moenomycin production, in which the C15-hydrocarbon moiety of farnesyl pyrophosphate (FPP) is transferred to the 2-hydroxy group of 3-phosphoglycerate (3PG), forming an ether bond (Scheme 1).[2] The reaction is similar to that catalyzed by geranylgeranylglyceryl phosphate synthase (GGGPS) for synthesizing archaea-type phospholipids.[3] However, in contrast to the enzyme GGGPS, which gives products with retained all-trans configuration of the isoprenyl chain, the enzyme MoeO5 leads to a trans-to-cis isomerization at the C2= C3 double bond of the transferred farnesyl group.[4] The crystal structures of the proteins GGGPS and the bacterial homologue PcrB reveal a triosephosphate isomerase (TIM)-barrel fold, which had not been observed previously in prenyltransferases.[3, 5] The sequences of GGGPS and PcrB share 35% amino acid identity, but MoeO5 shares only 10% identity with both enzymes (Figure S1 in the Supporting Information). Herein we report the X-ray crystallographic structures of MoeO5 bound to the product 2-(Z, E)-farnesyl-3-phosphoglycerate (FPG), to the substrate analogue farnesyl thiopyrophosphate (FsPP), and to magnesium (Mg2+) and pyrophosphate (PPi); together with additional biochemical and bioinformatics results, these structures shed light on the possible mechanisms of action of this unusual enzyme.Molecular-replacement approaches to determine the structure of MoeO5 by using GGGPS and PcrB as search models were not successful, thereby reflecting perhaps the significant variations in the protein sequences. Because MoeO5 contains no Cys residue, to solve the structure by using multiple isomorphous replacement (MIR), we produced the mutant H97C for efficient preparation of mercurybased MIR derivatives (Table S1 in the Supporting Information). The other structures were solved by molecular replacement (Figure S2 in the Supporting Information and Table 1; see the Supporting Information for details). MoeO5 crystallizes as a homodimer (FigureS3 and TableS2 in the Supporting Information). The dimer interface buries 1200 2, which is more than 10% surface area, on each monomer and mainly involves hydrophobic residues in helices a4 and a5. The cis-peptide of Phe140–Pro141 binds to a Mg2+ at the molecular dyad (Figure S3 in the Supporting Information). These helices also mediate dimerization in GGGPS and PcrB, but in MoeO5 one of the TIM barrels is rotated by 1808 (Figure S4 in the Supporting Information). A more detailed description of the protein structure as well as of the bound ligands can be found in the Supporting Information. Despite its Ca root-mean-square deviation (rmsd) of about 2.0 from the GGGPS and PcrB monomers (Figure S5 in the Supporting Information), the similar protein fold with a connecting loop (denoted l3) between strands b3 and b4 clearly places MoeO5 among this new class of TIM-barrel prenyltransferases.
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影响因子：
15
作者：
Doud EH;Perlstein DL;Wolpert M;Cane DE;Walker S
通讯作者：
Walker S
影响因子：
2.9
作者：
Ostash B;Doud EH;Lin C;Ostash I;Perlstein DL;Fuse S;Wolpert M;Kahne D;Walker S
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Walker S
影响因子：
2.9
作者：
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Liang, Po-Huang
影响因子：
4.8
作者：
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通讯作者：
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影响因子：
16.6
作者：
Guldan, Harald;Matysik, Frank-Michael;Babinger, Patrick
通讯作者：
Babinger, Patrick