Hupert-Kocurek Katarzyna, Wojcieszyńska Danuta, Guzik Urszula
Department of Biochemistry, Faculty of Biology and Environmental Protection, University of Silesia in Katowice, Katowice, Poland.
Acta Biochim Pol. 2014;61(4):705-10. Epub 2014 Oct 22.
c23o gene, encoding catechol 2,3-dioxygenase from Planococcus sp. strain S5 was randomly mutagenized to generate variant forms of the enzyme with higher degradation activity. Additionally, the effect of introduced mutations on the enzyme structure was analyzed based on the putative 3D models the wild-type and mutant enzymes. C23OB58 and C23OB81 mutant proteins with amino acid substitutions in close proximity to the enzyme surface or at the interface and in the vicinity of the enzyme active site respectively showed the lowest activity towards all catecholic substrates. The relative activity of C23OC61 mutant towards para-substituted catechols was 20-30% lower of the wild-type enzyme. In this mutant all changes: F191I, C268R, Y272H, V280A and Y293D were located within the conserved regions of C-terminal domain. From these F191I seems to have significant implications for enzyme activity. The highest activity towards different catechols was found for mutant C23OB65. R296Q mutation improved the activity of C23O especially against 4-chlorocatechol. The relative activity of above-mentioned mutant detected against this substrate was almost 6-fold higher than the wild-type enzyme. These results should facilitate future engineering of the enzyme for bioremediation.
编码来自嗜皮菌属菌株S5的儿茶酚2,3 -双加氧酶的c23o基因被随机诱变，以产生具有更高降解活性的酶变体形式。此外，基于野生型和突变型酶的推测三维模型，分析了引入的突变对酶结构的影响。分别在靠近酶表面或界面以及酶活性位点附近具有氨基酸取代的C23OB58和C23OB81突变蛋白对所有儿茶酚底物的活性最低。C23OC61突变体对对位取代儿茶酚的相对活性比野生型酶低20 - 30%。在这个突变体中，所有变化：F191I、C268R、Y272H、V280A和Y293D都位于C末端结构域的保守区域内。其中F191I似乎对酶活性有重大影响。发现突变体C23OB65对不同儿茶酚的活性最高。R296Q突变提高了C23O的活性，尤其是对4 -氯儿茶酚的活性。针对该底物检测到的上述突变体的相对活性比野生型酶高出近6倍。这些结果应有助于该酶未来用于生物修复的工程改造。
