Structure-function relationships of the lanthionine cyclase SpaC involved in biosynthesis of the Bacillus subtilis peptide antibiotic subtilin
Structure-function relationships of the lanthionine cyclase SpaC involved in biosynthesis of the Bacillus subtilis peptide antibiotic subtilin
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DOI：
10.1021/bi062124f
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发表时间：
2007-03-20
期刊：
影响因子：
2.9
通讯作者：
Stein, Torsten
中科院分区：
文献类型：
--
作者：
Helfrich, Markus;Entian, Karl-Dieter;Stein, Torsten
在枯草芽孢杆菌中，羊毛硫抗生素枯草菌素的生物合成是通过合成酶复合物完成的，所述合成酶复合物由乙酰化酶SpaB、环化酶SpaC和转运蛋白SpaT组成。基因工程枯草杆菌环化酶SpaC和相关的NisC和EriC蛋白参与的羊毛硫抗生素乳链菌肽和ericin A/S的生物合成，分别进行了分析，功能上取代天然SpaC在体内。我们可以显示的第一次lantibiotic前体肽（枯草菌素）的翻译后修饰的混合lantibiotic合成酶（SpaBT/EriC）。基因工程SpaC丙氨酸置换突变体揭示了残基His（231）、Trp（302）、Cys（303）、Tyr（304）、Gly（305）、Cys（349）和His（350）以及保守的C端基序Lys（437）-Ala（438）-Leu（439）-Leu（440）-Ile（441）对于枯草杆菌素生物合成的必要性。将这些严格保守的羊毛硫抗生素环化酶残基指定为NisC结构[Li，B.，Yu，J. B.，Brunzelle，J.S.，Moll，G. N.，货车德东克，W.一、和Nair，S. K.（2006）Science，311，1464-1467]揭示了羊毛硫抗生素环化酶的催化中心中的结构-功能关系的第一个实验证据。SpaC残基His（231）、Cys（303）和Cys（349）参与中心锌离子的配位。讨论了His（231）/Tyr（304）对作为镧系元素形成中的一般酸/碱催化剂。此外，下拉实验表明，功能失活SpaC突变体仍然能够在体外与六组氨酸标记的枯草杆菌素前体肽相互作用。我们的研究结果表明，色氨酸（302）和SpaC的C-末端残基的疏水簇，这是参与稳定的催化中心和结合的枯草杆菌素前体肽的成分。
Biosynthesis of the lantibiotic subtilin in Bacillus subtilis is accomplished by a synthetase complex consisting of the dehydratase SpaB, cyclase SpaC, and transporter SpaT. Genetically engineered subtilin cyclases SpaC and related NisC and EriC proteins involved in biosynthesis of the lantibiotics nisin and ericin A/S, respectively, were analyzed to functionally substitute native SpaC in vivo. We could show for the first time posttranslational modification of a lantibiotic precursor peptide (subtilin) by a hybrid lantibiotic synthetase (SpaBT/EriC). Genetically engineered SpaC alanine replacement mutants revealed the essentiality of residues His(231), Trp(302), Cys(303), Tyr(304), Gly(305), Cys(349), and His(350), as well as the conserved C-terminal motif Lys(437)-Ala(438)-Leu(439)-Leu(440)-Ile(441) for subtilin biosynthesis. Assignment of these strictly conserved lantibiotic cyclase residues to the NisC structure [Li, B., Yu, J. B., Brunzelle, J. S., Moll, G. N., van der Donk, W. A., and Nair, S. K. (2006) Science, 311, 1464-1467] revealed the first experimental evidence for structure-function relationships in catalytic centers of lantibiotic cyclases. SpaC residues His(231), Cys(303), and Cys(349) are involved in coordination of the central zinc ion. The pair His(231)/Tyr(304) is discussed to act as general acid/base catalysts in lanthionine formation. Furthermore, pull-down experiments revealed that functional inactive SpaC mutants were still able to interact with the hexahistidine-tagged subtilin precursor peptide in vitro. Our results suggest that Trp(302) and the C-terminal residues of SpaC are constituents of a hydrophobic cluster which is involved in stabilization of the catalytic center and binding of the subtilin precursor peptide.