Mechanism Of D-malate Metabolism In Pseudomonas And Regulation Of D-2-hydroxyglutarate Metabolism In Achromobacter

Glycolysis and TCA cycle are two core metabolic pathways in organisms.Pyruvate,oxaloacetate,and 2-ketoglutarate(2-KG)are three key intermediates that belong to 2-keto carboxylic acids in glycolysis and TCA.In the presence of reducing agents and reductases,these 2-keto carboxylic acids can be reduced into lactate,malate,and 2-hydroxyglutarate(2-HG),respectively.The mechanism of lactate metabolism has been well-studied in different organisms,while relatively few studies have focused on D-2-HG and D-malate.In recent years,it was found that various kinds of cancers are accompanied with D-2-HG accumulation and D-2-HG could serve as a toxic metabolic product to promote oncogenesis.Recently,we found that D-2-HG from Pseudomonas stutzeri A1501 is an important hidden metabolite produced by D-3-phosphoglycerate dehydrogenase(SerA).Coupling of D-3-phosphoglycerate(D-3-PG)and D-2-HG metabolism overcomes the thermodynamic barrier of D-3-PG dehydrogenation to 3-phosphohydroxypyruvate(3-PHP)and promotes the biosynthesis of L-serine.D-Malate is a four carbon dicarboxylic acid that is similar to the structure of D-2-HG.It has been reported that various species have the ability to utilize D-malate for growth.D-Malate dehydrogenase(DmlA)from Escherichia coli has been reported to oxidatively decarboxylate D-malate to pyruvate,but the homologous protein of DmlA has not been found in Pseudomonas.This paper found that SerA from P.stutzeri A1501 can reduce oxaloacetate to D-malate and catalyze the reduction of 2-KG to D-2-HG simultaneously.D-2-HG dehydrogenase(D2HGDH)in P.stutzeri A1501 has the activity of catalyzing the dehydrogenation of D-malate.D2HGDH can convert D-2-HG to 2-KG and convert D-malate to oxaloacetate during L-serine biosynthesis.In addition,D2HGDH is also a key enzyme for the growth of P.stutzeri A1501 using D-malate as the sole carbon source.D2HGDH plays a dual role in the extracellular D-malate utilization and the core metabolic pathway of L-serine synthesis.Exogenous D-2-HG and D-malate have the ability to induce the expression of D2HGDH in P.stutzeri A1501,but the related regulatory protein was not discovered upstream or downstream of the gene d2hgdh.Through bioinformatics analysis,this paper found that there is d2hgdh gene in the genome of Achromobacter xylosoxidans ATCC 27061 and Achromobacter denitrificans NBRC 15125(ATCC 15173),which is adjacent to the regulatory protein of GntR family.We selected A.denitrificans NBRC 15125 as the research object,and found that DhdR is the key regulator regulating D2HGDH expression.DhdR plays a repressive role in the transcription of d2hgdh gene.Experiments such as qRT-PCR,EMSA and enzyme activity analysis demonstrated that DhdR regulates D2HGDH expression by specific response to D-2-HG.