无色杆菌中D-2-羟基戊二酸脱氢酶及其调控机制研究

D-2-Hydroxyglutarate dehydrogenase (D2HGDH) plays a critical role in D-2-hydroxyglutarate catabolism that is important for organisms. We have reported that the coupling between D-3-phosphoglycerate dehydrogenase (SerA) and D2HGDH drives L-serine synthesis in Pseudomonas. The cellular demand for L-serine keeps changing under various environmental and physiological conditions. Thus, the function of SerA and D2HGDH needs to be coordinated during the coupling. However, the mechanism is unknown. In additon, the distribution of D2HGDH is essential to analyzing bacterial D-2-HG metabolism. Based on the comparative genomic analysis and preliminary experimental results, we found that homologs of D2HGDH and SerA coexists in Achromobacter. Interestingly, the gene encoding a regulatory protein of GntR family (DhdR) is adjacent to the D2HGDH-encoding gene, suggesting the roles of DhdR in regulation of D2HGDH expression. In this project, the methods including bioinformatics, molecular physiology and biochemistry studies would be employed. The biological function of the D2HGDH homolog in A. denitrificans would be verified, and the distribution of D2HGDH in bacteria would be further analyzed. We would also identify DhdR as the key transcriptional regulation factor for D2HGDH, and clarify the molecular mechanism to regulate D2HGDH expression. Then, after the demonstration for the coupling module between SerA and D2HGDH to drive L-serine biosynthesis in A. denitrificans, the roles of DhdR in coordinating the function and operation of the coupling would be explored. The uncovered regulatory mechanism of D-2-HG metabolism in A. denitrificans through the accomplishment of the project would be of significance for D-2-HG metabolism and L-serine synthesis of organisms.

D-2-羟基戊二酸脱氢酶（D2HGDH）参与生物体中重要的D-2-羟基戊二酸（D-2-HG）分解代谢。申请者已报道假单胞菌D2HGDH与磷酸甘油酸脱氢酶（SerA）耦合驱动了丝氨酸合成，但其中两酶功能协调机制亟待阐明，同时D2HGDH在细菌中分布亦需明确。申请人基于比较基因组学分析与前期实验结果，发现无色杆菌属细菌中存在D2HGDH和SerA同源蛋白及GntR家族转录调控蛋白（DhdR）。本项目拟基于上述关键蛋白生物信息学、生理生化及分子作用分析，验证D2HGDH同源蛋白生物学功能，分析细菌中D2HGDH分布广度；确定DhdR为调控D2HGDH表达的关键因子，并阐明分子调控机理；证明无色杆菌中同样存在SerA和D2HGDH耦合驱动丝氨酸合成的代谢模块，探究DhdR参与其中协调运行的功能角色，揭示D-2-HG代谢调控机制。本研究对于完善生物体D-2-HG代谢和丝氨酸合成机制有重要意义。

D-2-羟基戊二酸脱氢酶（D2HGDH）参与生物体中重要的D-2-羟基戊二酸（D-2-HG）分解代谢。项目团队之前已报道假单胞菌D2HGDH与磷酸甘油酸脱氢酶（SerA）耦合驱动了丝氨酸合成。假单胞菌D2HGDH在细菌中的同源蛋白与其一致性主要在30%—40%之间，阐明这些同源蛋白的生化功能是D2HGDH，有助于进一步确定D-2-HG代谢在细菌中分布的广泛性。另外，D-2-HG代谢中D2HGDH表达调控机制目前尚未得到阐明。Achromobacter denitrificans NBRC 15125中存在D2HGDH同源蛋白、SerA，以及在D2HGDH同源蛋白编码基因上游的GntR家族转录调控蛋白（DhdR），因此该菌株是研究D-2-HG代谢调控机制的优秀模式生物。项目以其为研究对象，主要探究该菌株中D2HGDH表达调控机制及D-2-HG代谢模型。本项目的顺利开展，成功地纯化了该菌株中D2HGDH，表征了其生化功能，证明其为D-2-HG分解代谢关键酶，提示了D2HGDH在细菌中分布的广泛性；纯化并表征了DhdR蛋白，确定了DhdR为D2HGDH表达的关键阻遏调控因子，发现了DhdR和D2HGDH共转录，鉴定了转录起始位点及DhdR的关键结合DNA序列，证明了D-2-HG是DhdR的特异性效应物；纯化了该菌株中SerA，发现其能够体外催化还原2-酮基戊二酸产生D-2-HG，但在生理状况下并不负责胞内D-2-HG合成代谢，其功能缺失也不能造成丝氨酸营养缺陷型，即该菌株中SerA并不能与D2HGDH形成代谢耦合；得到了D-2-HG代谢模型，即菌体吸收外源的D-2-HG，特性性调控因子DhdR感知D-2-HG浓度的变化，调控D2HGDH的表达，进而代谢并同化D-2-HG。另外，团队比较了不同生物体来源的D2HGDH的底物谱，发现无色杆菌来源的D2HGDH具有最好的底物特异性，借此探索了针对肿瘤标志物D-2-HG的检测方法。本项目对A. denitrificans NBRC 15125中D-2-HG代谢调控开展研究，得到关于微生物中D-2-HG代谢机制的新认识，为深入揭示生物体内D-2-HG代谢相关生理过程提供有益借鉴和理论基础。同时，项目获得的特异性调控蛋白DhdR和D2HGDH，有潜力开发D-2-HG生物传感器及肿瘤诊疗基因线路。