The NADH recycling enzymes TsaC and TsaD regenerate reducing equivalents for Rieske oxygenase chemistry.
The NADH recycling enzymes TsaC and TsaD regenerate reducing equivalents for Rieske oxygenase chemistry.
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NADH回收酶TSAC和TSAD再生rieske oxygygoase Chemistry降低等效物。
DOI：
10.1016/j.jbc.2023.105222
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发表时间：
2023-10
影响因子：
4.8
通讯作者：
Bridwell-Rabb, Jennifer
中科院分区：
文献类型：
--
作者：
Tian, Jiayi;Boggs, David G.;Donnan, Patrick H.;Barroso, Gage T.;Garcia, Alejandro Arcadio;Dowling, Daniel P.;Buss, Joshua A.;Bridwell-Rabb, Jennifer
许多微生物使用生物和非生物分子作为碳和能量的来源。这种资源意味着一些微生物具有吸收环境中发现的污染物的机制。一种这样的生物体是睾丸酮丛毛单胞菌，其使用TsaMBCD途径代谢4-甲基苯磺酸盐和4-甲基苯甲酸盐。TsaM是Rieske加氧酶，其与还原酶TsaB协同消耗摩尔当量的NADH。在该步骤之后，注释的短链脱氢酶/还原酶和醛脱氢酶TsaC和TsaD各自再生摩尔当量的NADH。这种共存改善了化学计量添加还原当量的需要，因此代表了将Rieske加氧酶化学整合到生物催化应用中的有吸引力的策略。因此，在这项工作中，为了克服缺乏有关与Rieske非血红素铁加氧酶（Rieske加氧酶）合作发挥作用的NADH再循环酶的信息，我们将TsaC的X射线晶体结构解析为2.18 nm。使用这种结构，一系列的底物类似物和蛋白质变体组合反应，和差示扫描荧光实验，我们确定了参与结合NAD+和控制底物特异性的活性位点特征。进一步的体外酶级联实验证明了有效的TsaC和TsaD介导的NADH再生，以支持Rieske加氧酶化学。最后，通过深入的生物信息学分析，我们说明了广泛共存的Rieske加氧酶与TsaC样酶。因此，这项工作证明了这些NADH回收酶的效用，并确定了一个库的短链脱氢酶/还原酶的前景，可用于Rieske加氧酶途径的原位再生的NADH。
Many microorganisms use both biological and nonbiological molecules as sources of carbon and energy. This resourcefulness means that some microorganisms have mechanisms to assimilate pollutants found in the environment. One such organism is Comamonas testosteroni, which metabolizes 4-methylbenzenesulfonate and 4-methylbenzoate using the TsaMBCD pathway. TsaM is a Rieske oxygenase, which in concert with the reductase TsaB consumes a molar equivalent of NADH. Following this step, the annotated short-chain dehydrogenase/reductase and aldehyde dehydrogenase enzymes TsaC and TsaD each regenerate a molar equivalent of NADH. This co-occurrence ameliorates the need for stoichiometric addition of reducing equivalents and thus represents an attractive strategy for integration of Rieske oxygenase chemistry into biocatalytic applications. Therefore, in this work, to overcome the lack of information regarding NADH recycling enzymes that function in partnership with Rieske non-heme iron oxygenases (Rieske oxygenases), we solved the X-ray crystal structure of TsaC to a resolution of 2.18 Å. Using this structure, a series of substrate analog and protein variant combination reactions, and differential scanning fluorimetry experiments, we identified active site features involved in binding NAD+ and controlling substrate specificity. Further in vitro enzyme cascade experiments demonstrated the efficient TsaC- and TsaD-mediated regeneration of NADH to support Rieske oxygenase chemistry. Finally, through in-depth bioinformatic analyses, we illustrate the widespread co-occurrence of Rieske oxygenases with TsaC-like enzymes. This work thus demonstrates the utility of these NADH recycling enzymes and identifies a library of short-chain dehydrogenase/reductase enzyme prospects that can be used in Rieske oxygenase pathways for in situ regeneration of NADH.
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影响因子：
14.9
作者：
Blum M;Chang HY;Chuguransky S;Grego T;Kandasaamy S;Mitchell A;Nuka G;Paysan-Lafosse T;Qureshi M;Raj S;Richardson L;Salazar GA;Williams L;Bork P;Bridge A;Gough J;Haft DH;Letunic I;Marchler-Bauer A;Mi H;Natale DA;Necci M;Orengo CA;Pandurangan AP;Rivoire C;Sigrist CJA;Sillitoe I;Thanki N;Thomas PD;Tosatto SCE;Wu CH;Bateman A;Finn RD
通讯作者：
Finn RD
影响因子：
5.6
作者：
Dumitru, Razvan;Jiang, Wen Zhi;Weeks, Donald P.;Wilson, Mark A.
通讯作者：
Wilson, Mark A.
DOI：
10.1107/s0907444905036693
发表时间：
2006-01-01
影响因子：
2.2
作者：
Evans, P
通讯作者：
Evans, P
影响因子：
4.8
作者：
Mattice, Jenna R.;Shisler, Krista A.;DuBois, Jennifer L.;Peters, John W.;Bothner, Brian
通讯作者：
Bothner, Brian
影响因子：
56.9
作者：
CORRELL, CC;BATIE, CJ;LUDWIG, ML
通讯作者：
LUDWIG, ML