Identification Of Key Enzymes Involved In Aromatic Compounds Catabolism Via Gentisate Pathway By Pseudomonas Putida XL501 And Haloferax Vocalnii WFD11

Aromatic compounds are a class of chemicals with one or more benzene rings.The aromatic compounds degradation via gentisate(2,5-dihydroxybenzoate)pathway have been reported in a number of bacteria and fungi.However,there are limited reports about aromatics degradation via gentisate by archaea.The gentisate pathway,with isomerization and hydrolysis branches,plays an important role in the central metabolism of microbial aromatic compounds degradation.In bacteria,Pseudomonas putida NCIMB 9869 was the sole one which have the above two branches of gentisate pathway,but no reports at the genetic and molecular level for this interesting phenomenon.On the other hand,only Haloferax sp.strain D1227 and Haloarcula sp.strain D1 were reported to degrade aromatics via gentisate pathway among the halophilic archaea,but there was no molecular and biochemical studies reports on this complete pathway.In this study,the genome sequence of P.putida XL501(the spontaneous mutant strain of P.putida NCIMB 9869)was analyzed by bioinformatics.Three putative key enzymes-encoding genes of gentisate pathway were found: the putative maleylpyruvate isomerase-encoding gene xlgL and two putative maleylpyruvate hydrolase-encoding genes xlgF1 and xlgF2.The above three genes were all overexpressed in E.coli BL21(DE3)and the enzymes expressed were purified..The cell extract of E.coli BL21(DE3)[pET-28a-xlgL] as well as the purified XlgL exhibited maleylpyruvate isomerase activity with specific activities of 0.0003 0.0003 and 0.0035 0.0011 U/mg,respectively.Meanwhile,the cell extract of E.coli BL21(DE3)[pET-28a-xlgF1] and E.coli BL21(DE3)[pET-28a-xlgF2] both showed maleylpyruvate hydrolase activity,with specific activities of 0.0028 0.0032 and 0.0018 0.0006U/mg,respectively.In addition,purified XlgF1 and XlgF2 also exhibited the maleylpyruvate hydrolase activity with specific activities of 0.1700 0.0300 and 0.1500 0.0458 U/mg,respectively.Haloferax vocalnii WFD11 is commonly used as the expression host of halophilic archaea rather than an aromatic compounds utilizer.In this study,through high performance liquid chromatography(HPLC),we found that gentisate was the intermediate during 3HBA catabolism by strain WFD11.By bioinformatic analysis,the putative gentisate 1,2-dioxygenase-encoding gene hagA was found.When hagA was expressed in H.vocalnii H1424,the cell extract of H.vocalnii H1424[pTA1288-hagA] and the purified HagA both exhibited the activity of gentisate 1,2-dioxygenase with the specific activities of 0.0100 and 0.0248 U/mg,respectively.In addition,through Real-time PCR,it was proved that hagA was constitutively transcribed in strain WFD11.This study further illustrates the diversity of gentisate pathways in bacterial aromatic degradation,and also help us understand the possible differences between bacteria and archaea in aromatic compounds degradation via gentisate.