Structural and functional characterization of BaiA, an enzyme involved in secondary bile acid synthesis in human gut microbe.
Structural and functional characterization of BaiA, an enzyme involved in secondary bile acid synthesis in human gut microbe.
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DOI：
10.1002/prot.24353
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发表时间：
2014-02
影响因子：
2.9
通讯作者：
Lesley, Scott A.
中科院分区：
文献类型：
--
作者：
Bhowmik, Shiva;Jones, David H.;Chiu, Hsien-Po;Park, In-Hee;Chiu, Hsiu-Ju;Axelrod, Herbert L.;Farr, Carol L.;Tien, Henry J.;Agarwalla, Sanjay;Lesley, Scott A.
尽管次级胆汁酸对人类健康和疾病有重要影响，但催化其合成的关键肠道微生物酶的结构和生化信息有限。在此，我们报告载脂蛋白和辅因子结合的晶体结构的BaiA 2，短链脱氢酶/还原酶从梭菌VPI 12708，代表第一个蛋白质结构的这一途径。这些结构阐明了辅因子特异性的基础和质子传递的机制。涉及Glu 42位于辅因子结合位点的构象限制似乎是决定辅因子特异性的关键。Glu 42的有限柔性导致与NADP（H）的2′-磷酸基团的空间位阻和静电位阻。与晶体结构一致，用BaiA 2和BaiA 1（具有92%序列同一性的紧密同源物）进行的稳态动力学表征揭示NADP+的特异性常数（kcat/KM）比NAD+低至少一个数量级。与野生型相比，用Ala取代Glu 42将对NADP+的特异性提高了10倍。辅因子结合结构揭示了一种新的烟酰胺-氢氧根离子（NAD+-OH−）加合物，与先前报道的加合物相反。BaiA 2中加合物的OH−位于烟酰胺的C4原子的远端，核糖部分的2′-羟基的近端。此外，它位于活性位点残基Tyr 157（2.7 nm）和Lys 161（4.5 nm）的末端官能团之间的中间距离处。基于这些观察结果，我们提出NAD+-OH−加合物参与质子传递，而不是之前加合物中提到的氢化物转移。
Despite significant influence of secondary bile acids on human health and disease, limited structural and biochemical information is available for the key gut microbial enzymes catalyzing its synthesis. Herein, we report apo- and co-factor bound crystal structures of BaiA2, a short chain dehydrogenase/reductase from Clostridium scindens VPI 12708 that represent the first protein structure of this pathway. The structures elucidated the basis of co-factor specificity and mechanism of proton relay. A conformational restriction involving Glu42 located in the co-factor binding site seems crucial in determining co-factor specificity. Limited flexibility of Glu42 results in imminent steric and electrostatic hindrance with 2′-phosphate group of NADP(H). Consistent with crystal structures, steady-state kinetic characterization performed with both BaiA2 and BaiA1, a close homolog with 92% sequence identity, revealed specificity constant (kcat/KM) of NADP+ at least an order of magnitude lower than NAD+. Substitution of Glu42 with Ala improved specificity towards NADP+ by 10- fold compared to wild type. The co-factor bound structure uncovered a novel nicotinamide-hydroxyl ion (NAD+-OH−) adduct contraposing previously reported adducts. The OH− of the adduct in BaiA2 is distal to C4 atom of nicotinamide and proximal to 2′-hydroxyl group of the ribose moiety. Moreover, it is located at intermediary distances between terminal functional groups of active site residues Tyr157 (2.7 Å) and Lys161 (4.5 Å). Based on these observations we propose an involvement of NAD+-OH− adduct in proton relay instead of hydride transfer as noted for previous adducts.
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影响因子：
64.8
作者：
通讯作者：
--
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
DOI：
10.1107/s1744309110038212
发表时间：
2010-10-01
影响因子：
0.9
作者：
Elsliger, Marc-Andre;Deacon, Ashley M.;Wilson, Ian A.
通讯作者：
Wilson, Ian A.
影响因子：
2.6
作者：
MALLONEE, DH;LIJEWSKI, MA;HYLEMON, PB
通讯作者：
HYLEMON, PB
影响因子：
2.9
作者：
Klock, Heath E.;Koesema, Eric J.;Lesley, Scott A.
通讯作者：
Lesley, Scott A.