Biosynthesis Of Coenzyme Q In Xanthomonas Campestris Pv.campestris

Coenzyme Q(Co Q)is a widespread redox-active quinone compound.As the electron and proton carrier,Co Q is essential for aerobic respiration and energy generation in eukaryotes and prokaryotes.In this study,the Co Q biosynthesis in Xanthomonas campestris pv.campestris(Xcc)was investigated.Firstly,using Escherichia coli 11 ubi genes as templates,BLASTp and domain analysis identified 7 Xcc homologous genes for coenzyme Q biosynthesis,including Xc?0402(ubi A),Xc?0234(ubi B),Xc?0673(ubi E),Xc?1846(ubi G),Xc?3432(ubi H),Xc?3433(ubi I),Xc?0233(ubi J).Xcc genome lacks the homologous genes for ubi C,ubi D/X and ubi F.Our previous work has revealed that Xan B2,a unique chorismate lyase with no homology to Ubi C,is responsible for 4-hydroxybenzoate production in Xcc Co Q biosynthesis.Secondly,in order to search for Xcc ubi D/X homologous genes,a genomic library of Xcc strain 8004 was constructed.The plasmids from the constructed library were introduced into E.coli MG1655?ubi D cells by electroporation(ET)and heat shock(HT),respectively.Ten of them were found to increase the growth of E.coli MG1655?ubi D cells.However,none of them was found to be involved in Co Q biosynthesis in Xcc.Tirdly,by screening the mutant library constructed in Xcc strain 8004,which was constructed by Guang Xi University,we found that mutation in Xc?0489 resulted in a dramatic decrease in Co Q content.An unknown compound X could be detected in?Xc?0489 cells.Single copy integration of the Xc?0489 ORF and its cognate promoter in the chromosome of(35)Xc?0489 cells restored Co Q content and abolished the accumulation of compound X.Analysis by high resolution spectrometry in positive mode gave a m/z ratio(M+H⁺)of 719.5369 for compound X to chemical formula of C₄₈H₇₂O₃.This is very close to the molecular weight of DMQ₈,one of the metabolic intermediates in Co Q₈ biosynthetic pathway.To further demonstrate its true biological function,we conducted the interspecific complementation analysis of E.coli ubi F and Xc?0489.E.coli C6-hydroxylase gene ubi F completely complement?Xc?0489.Xc?0489was also able to partially complement E.coli?ubi F.The data suggests that Xc?0489 encodes C6-hydroxylase in Co Q biosynthesis.Consequently,Xc?0489 was renamed as“xbi F”in this study.Domain analysis showed that Xbi F does not belong to the“Ubi I/Ubi H/Ubi F/COQ6 hydroxylase superfamily”in bacterial Co Q biosynthesis,but constitutes the“Ferritin?like superfamily”together with Saccharomyces cerevisiae COQ7 and Caenorhabditis elegans CLK-1.Further point mutation and protein modeling experiments suggest a putative three-dimensional structure and catalytic mechanism of Xbi F.In conclusion,the present study demonstrated the biological function of Xc?0489 in Xcc Co Q biosynthesis.Since Co Q is essential for Xcc survival and virulence,the biosynthetic pathway may provide a potential target for designing green pesticide to prevent this phytopathogen.