Catalytic properties of the expressed acyclic carotenoid 2-ketolases from Rhodobacter capsulatus and Rubrivivax gelatinosus
Catalytic properties of the expressed acyclic carotenoid 2-ketolases from Rhodobacter capsulatus and Rubrivivax gelatinosus
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DOI：
10.1016/j.bbalip.2008.12.006
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发表时间：
2009-02-01
影响因子：
4.8
通讯作者：
Sandmann, Gerhard
中科院分区：
文献类型：
--
作者：
Gerjets, Tanja;Steiger, Sabine;Sandmann, Gerhard
紫色光合细菌在有氧生长条件下可合成无环类胡萝卜素球甾烯和螺旋黄素，酮化产物为球甾酮和2,2′-二酮螺旋黄素。为了研究酮化酶的催化反应，在大肠杆菌中将明胶红杆菌和荚膜红杆菌编码无环类胡萝卜素2-酮化酶的crtA基因表达为功能酶。用后者的纯化酶，测定了分子氧和还原铁氧还蛋白对催化活性的要求。此外，假定的中间产物2- ho -球烯在体外转化为相应的2-酮产物。因此，提出了该酶的单加氧酶机制，涉及C-2上两个连续的羟基化步骤。通过大肠杆菌的功能途径互补研究和酶动力学研究，对两种酶的产物特异性进行了研究。酮酶可以催化球黄质和螺旋黄质途径的大部分中间体和产物。来自Rba的酶也是如此。没有螺旋黄素合成的荚膜。一般来说，Rvi的酮醇酶。gelatinosus对球质、ho -球质和螺旋黄质作为底物的特异性优于Rba中的酮醇酶。capsulatus。(C) 2008 Elsevier B.V.版权所有
Purple photosynthetic bacteria synthesize the acyclic carotenoids spheroidene and spirilloxanthin which are ketolated to spheroidenone and 2,2'-diketospirilloxanthin under aerobic growth. For the studies of the catalytic reaction of the ketolating enzyme, the crtA genes from Rubrivivax gelatinosus and Rhodobacter capsulatus encoding acyclic carotenoid 2-ketolases were expressed in Escherichia coli to functional enzymes. With the purified enzyme from the latter, the requirement of molecular oxygen and reduced ferredoxin for the catalytic activity was determined. Furthermore, the putative intermediate 2-HO-spheroidene was in vitro converted to the corresponding 2-keto product. Therefore, a monooxygenase mechanism involving two consecutive hydroxylation steps at C-2 were proposed for this enzyme. By functional pathway complementation studies in E coli and enzyme kinetic studies, the product specificity of both enzymes were investigated. it appears that the ketolases could catalyze most intermediates and products of the spheroidene and spirilloxanthin pathway. This was also the case for the enzyme from Rba. capsulatus from which spirilloxanthin synthesis is absent. In general, the ketolase of Rvi. gelatinosus had a better specificity for spheroidene, HO-spheroidene and spirilloxanthin as substrates than the ketolase from Rba. capsulatus. (C) 2008 Elsevier B.V. All rights reserved.