Evidence for the Chemical Mechanism of RibB (3,4-Dihydroxy-2-butanone 4-phosphate Synthase) of Riboflavin Biosynthesis.
Evidence for the Chemical Mechanism of RibB (3,4-Dihydroxy-2-butanone 4-phosphate Synthase) of Riboflavin Biosynthesis.
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DOI：
10.1021/jacs.2c03376
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发表时间：
2022-07-20
影响因子：
15
通讯作者：
Lamb, Audrey L.
中科院分区：
文献类型：
--
作者：
Kenjic, Nikola;Meneely, Kathleen M.;Wherritt, Daniel J.;Denler, Melissa C.;Jackson, Timothy A.;Moran, Graham R.;Lamb, Audrey L.
RIBB(3，4-二羟基-2-丁酮-4-磷酸合成酶)是一种依赖镁的酶，它将d-核酮糖-5-磷酸(d-Ru5P)的C4分解为甲酸盐。RIBB为鲁马津合成酶产生四碳底物，该底物被结合到鲁马津的二甲苯部分中，并最终合成核黄素四氧嘧啶。Bacher和他的同事在20世纪90年代首次发现了这种反应，尽管直接证据很少，但他们的化学机制假说变得规范起来。当在非天然金属和/或配体非底物类似物存在下溶解时，Ribb的X射线晶体结构通常显示两个金属离子，并且一致的假设机制已经纳入了这个辅因子集。我们使用了各种生化方法来进一步表征霍乱弧菌Ribb(VcRibB)催化的化学作用。我们表明，当金属离子浓度等于酶浓度时，可以获得全部活性。重组酶的电子顺磁共振证实了这一点，该酶由锰和晶体结构与锰离子和多种糖磷酸盐连接而成。对单一和完全13C标记的d-Ru5P进行了单次翻转反应的酸猝灭结合核磁共振，鉴定了产物形成前的两个瞬时物种。这些数据表明，C1的脱水形成了第一个瞬时物种，它通过1，2迁移进行重排，将C5融合到C3，并生成水合C4，准备以甲酸盐的形式消除。从VcRibB-d-Ru5P晶体随时间变化的Mn2+浸泡确定的结构表明，在相同中间体的晶体中积累。总而言之，这些数据第一次揭示了RIBB机制中关键的瞬时化学状态。
RibB (3,4-dihydroxy-2-butanone 4-phosphate synthase) is a magnesium-dependent enzyme that excises the C4 of d-ribulose-5-phosphate (d-Ru5P) as formate. RibB generates the four-carbon substrate for lumazine synthase that is incorporated into the xylene moiety of lumazine and ultimately the riboflavin isoalloxazine. The reaction was first identified by Bacher and co-workers in the 1990s, and their chemical mechanism hypothesis became canonical despite minimal direct evidence. X-ray crystal structures of RibB typically show two metal ions when solved in the presence of non-native metals and/or liganding non-substrate analogues, and the consensus hypothetical mechanism has incorporated this cofactor set. We have used a variety of biochemical approaches to further characterize the chemistry catalyzed by RibB from Vibrio cholera (VcRibB). We show that full activity is achieved at metal ion concentrations equal to the enzyme concentration. This was confirmed by electron paramagnetic resonance of the enzyme reconstituted with manganese and crystal structures liganded with Mn2+ and a variety of sugar phosphates. Two transient species prior to the formation of products were identified using acid quench of single turnover reactions in combination with NMR for singly and fully 13C-labeled d-Ru5P. These data indicate that dehydration of C1 forms the first transient species, which undergoes rearrangement by a 1,2 migration, fusing C5 to C3 and generating a hydrated C4 that is poised for elimination as formate. Structures determined from time-dependent Mn2+ soaks of VcRibB-d-Ru5P crystals show accumulation in crystallo of the same intermediates. Collectively, these data reveal for the first time crucial transient chemical states in the mechanism of RibB.
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影响因子：
2.9
作者：
Kumar, Pankaj;Singh, Mirage;Karthikeyan, Subramanian
通讯作者：
Karthikeyan, Subramanian
影响因子：
5.7
作者：
Liao, DI;Calabrese, JC;Jordan, DB
通讯作者：
Jordan, DB
影响因子：
15
作者：
Antharavally, BS;Poyner, RR;Ludden, PW
通讯作者：
Ludden, PW
DOI：
10.1016/0304-4165(76)90127-6
发表时间：
1976-01-01
期刊：
BIOCHIMICA ET BIOPHYSICA ACTA
影响因子：
--
作者：
BRYN, K;STORMER, FC
通讯作者：
STORMER, FC
DOI：
10.1016/0020-711x(75)90125-1
发表时间：
1975-01-01
期刊：
INTERNATIONAL JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
JABASINI, MT;ALKHALIDI, UAS
通讯作者：
ALKHALIDI, UAS