Crystal structures of a 6-dimethylallyltryptophan synthase, IptA: Insights into substrate tolerance and enhancement of prenyltransferase activity
Crystal structures of a 6-dimethylallyltryptophan synthase, IptA: Insights into substrate tolerance and enhancement of prenyltransferase activity
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6-二甲基烯丙基色氨酸合酶 IptA 的晶体结构：深入了解底物耐受性和异戊二烯基转移酶活性的增强
DOI：
10.1016/j.bbrc.2022.01.018
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Shingo Nagano*
中科院分区：
文献类型：
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作者：
Hironori Suemune;Doukan Nishimura;Kenjiro Mizutani;Yusuke Sato;Tomoya Hino;Hiroshi Takagi;Yumi Shiozaki-Sato;Shunji Takahashi*;Shingo Nagano*
二甲基烯丙基色氨酸合成酶(DMATs)催化二甲基烯丙基焦磷酸(DMAPP)向吲哚环的转移反应。IPTA是DMATS家族的成员，参与链霉菌中6-二甲基烯丙基吲哚-3-甲醛的生物合成。SN-593，催化l-色氨酸的C6-戊烯基化反应。该酶表现出戊烯受体的混杂，并可以接受各种色氨酸衍生物，正如观察到的其他几个DMATS家族成员一样。尽管迄今已确定了DMATS的许多晶体结构，但底物混杂的结构基础以及对含吲哚的天然底物替代品的接受程度仍有待澄清。在本研究中，我们测定了三芳基-色氨酸衍生物(5-甲基，6-甲基和N-α-甲基-L-色氨酸)-DMSPP(二甲基烯丙基S-硫代吡咯磷酸酯；DMAPP的稳定类似物)-IPTA的晶体结构，以及无底物的IPTA和三芳基-Trp-DMSPP-IPTA的配合物的晶体结构。IPTA的整体结构呈现典型的ABBA折叠，这在DMATS家族成员中很常见，而EL-Trp和DMSPP则存在于ABBA桶内的隧道中。三芳基-Trp-DMSPP-酶复合体的晶体结构可以解释Glu84和His294辅助的l-Trp的C6原子上的亲电取代，这与以前对其他DMATs的研究结果相同。尽管1-色氨酸紧贴在活性中心口袋中，1-色氨酸周围的空位非常有限，但该酶通过底物吲哚环和活性中心相邻侧链的轻微移位来容纳5-甲基-和6-甲基-L-色氨酸，导致5-甲基-L-色氨酸和6-甲基-L-色氨酸的C7预烯基化活性较高。像许多其他DMATs一样，IPTA不能利用大于DMAPP的戊烯基供体。为了扩大戊烯基供体结合口袋，引入了W154A突变。不出所料，该突变株从色氨酸、香叶基和法尼基焦磷酸中产生了异戊烯基色氨酸。
Dimethylallyltryptophan synthases (DMATSs) catalyze the prenyl transfer reaction from dimethylallyl pyrophosphate (DMAPP) to an indole ring. IptA, a member of the DMATS family, is involved in biosynthesis of 6-dimethylallylindole-3-carbaldehyde inStreptomycessp. SN-593 and catalyzes the C6-prenylation ofl-Trp. The enzyme exhibits prenyl acceptor promiscuity and can accept various Trp derivatives, as observed in several other DMATS family members. Although many crystal structures of DMATS have been determined to date, the structural basis of substrate promiscuity and the acceptance of alternatives to indole-containing natural substrates remain to be clarified. In this study, we determined the crystal structures of the ternaryl-Trp derivative (5-methyl-, 6-methyl-, and Nα-methyl-l-Trp) -DMSPP (dimethylallylS-thiolopyrophosphate; stable analog of DMAPP) -enzyme complex of IptA, in addition to the substrate-free IptA and ternaryl-Trp-DMSPP-IptA complex crystal structures. The overall structure of IptA exhibited a typical ABBA-fold, which is commonly found in DMATS family members, whilel-Trp and DMSPP are found in a tunnel located inside the ABBA barrel. The crystal structure of the ternaryl-Trp-DMSPP-enzyme complex can explain the electrophilic substitution at the C6 atom ofl-Trp, which is assisted by Glu84 and His294, as previously suggested for other DMATSs. Althoughl-Trp snugly fitted into the active site pocket and the unoccupied space aroundl-Trp is very limited in thel-Trp-DMSPP-IptA complex structure, the enzyme can accommodate 5-methyl- and 6-methyl-l-Trp by slight relocation of the substrate indole ring and adjacent side chain in the active site, resulting in a higher prenylation activity for 5-methyl-l-Trp and C7 prenylation of 6-methyl-l-Trp. Like many other DMATSs, IptA cannot utilize prenyl donors larger than DMAPP. To enlarge the prenyl donor-binding pocket, the W154A mutation was introduced. As expected, this mutant produced prenylatedl-Trp froml-Trp and geranyl- and farnesyl pyrophosphate.