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Study of duplicated galU genes in Rhodococcus jostii and a putative new metabolic node for glucosamine-1P in rhodococci.
Biochimica et Biophysica Acta (BBA) - General Subjects ( IF 3 ) Pub Date : 2020-09-03 , DOI: 10.1016/j.bbagen.2020.129727
A E Cereijo 1 , M L Kuhn 2 , M A Hernández 3 , M A Ballicora 4 , A A Iglesias 1 , H M Alvarez 3 , M D Asencion Diez 1
Affiliation  

Backgound

Studying enzymes that determine glucose-1P fate in carbohydrate metabolism is important to better understand microorganisms as biotechnological tools. One example ripe for discovery is the UDP-glucose pyrophosphorylase enzyme from Rhodococcus spp. In the R. jostii genome, this gene is duplicated, whereas R. fascians contains only one copy.

Methods

We report the molecular cloning of galU genes from R. jostii and R. fascians to produce recombinant proteins RjoGalU1, RjoGalU2, and RfaGalU. Substrate saturation curves were conducted, kinetic parameters were obtained and the catalytic efficiency (kcat/Km) was used to analyze enzyme promiscuity. We also investigated the response of R. jostii GlmU pyrophosphorylase activity with different sugar-1Ps, which may compete for substrates with RjoGalU2.

Results

All enzymes were active as pyrophosphorylases and exhibited substrate promiscuity toward sugar-1Ps. Remarkably, RjoGalU2 exhibited one order of magnitude higher activity with glucosamine-1P than glucose-1P, the canonical substrate. Glucosamine-1P activity was also significant in RfaGalU. The efficient use of the phospho-amino-sugar suggests the feasibility of the reaction to occur in vivo. Also, RjoGalU2 and RfaGalU represent enzymatic tools for the production of (amino)glucosyl precursors for the putative synthesis of novel molecules.

Conclusions

Results support the hypothesis that partitioning of glucosamine-1P includes an uncharacterized metabolic node in Rhodococcus spp., which could be important for producing diverse alternatives for carbohydrate metabolism in biotechnological applications.

General significance

Results presented here provide a model to study evolutionary enzyme promiscuity, which could be used as a tool to expand an organism's metabolic repertoire by incorporating non-canonical substrates into novel metabolic pathways.



中文翻译:

研究红球菌中的galU基因重复,以及在红球菌中推定的氨基葡萄糖-1P的新代谢节点。

背景

研究确定碳水化合物代谢中葡萄糖-1P命运的酶对于更好地了解微生物作为生物技术工具非常重要。一个易于发现的例子是来自红球菌属的UDP-葡萄糖焦磷酸化酶。在R. jostii基因组中,此基因是重复的,而R. fascians仅包含一个拷贝。

方法

我们报告的分子克隆嘎鲁从基因R. jostiiR. fascians生产重组蛋白RJO GalU1,RJO GalU2和RFA嘎鲁。进行底物饱和曲线,获得动力学参数,并使用催化效率(k cat / K m)分析酶的混杂性。我们还调查了R. jostii GlmU焦磷酸化酶活性与不同糖-1P的反应,这些糖可能与Rjo GalU2竞争底物。

结果

所有的酶都具有焦磷酸酶的活性,并且对糖-1Ps表现出底物混杂。值得注意的是,Rjo GalU2对葡萄糖胺1P的活性比标准底物葡萄糖1P高一个数量级。氨基葡萄糖-1P活动也是在显著RFA嘎鲁。磷酸氨基糖的有效利用表明该反应在体内发生的可行性。此外,RJO GalU2和RFA嘎鲁表示为生产用于新分子的推定合成(氨基)葡糖前体酶的工具。

结论

结果支持以下假设:葡糖胺1P的分配在红球菌属中包括一个未表征的代谢节点,这对于在生物技术应用中产生碳水化合物代谢的多种替代方法可能很重要。

一般意义

此处提供的结果提供了一个研究进化酶混杂性的模型,该模型可通过将非规范性底物纳入新的代谢途径来用作扩展生物体代谢库的工具。

更新日期:2020-09-20
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