A Ferredoxin-dependent Dihydropyrimidine Dehydrogenase In Clostridium Chromiireducens

Pyrimidines are nitrogen-containing heterocyclic compound that are components of ribonucleic acid and deoxyribonucleic acid,and their precursor nucleotides and deoxyribonucleotides.Therefore,pyrimidines are essential to life,and are widespread in nature,and microorganisms use pyrimidine degradation to obtain carbon and nitrogen and energy.Three pyrimidine degradation pathways in bacteria have been discovered: the reductive pathway,Rut pathway and oxidative pathway.Among them,the most extensively studied is the reductive metabolic pathway.Dihydropyrimidine dehydrogenase(Pyd A)catalyzes the first step of the reductive pyrimidine degradation(Pyd)pathway in bacteria and eukaryotes.Pyd A homologs studied to date catalyze the reduction of uracil to dihydrouracil,coupled to the oxidation of NAD(P)H.Uracil reduction occurs at a flavin mononucleotide(FMN)site,and NAD(P)H oxidation occurs at a flavin adenine dinucleotide(FAD)site,with two ferredoxin(Fdx)domains thought to mediate inter-site electron transfer.In the investigation of Pyd pathway,we noticed that a Clostridial Pyd A homolog(Pyd Ac)lacked the FAD domain,and is present in a Pyd gene cluster in the strict anaerobic bacterium Clostridium chromiireducens.We therefore hypothesized that Pyd Ac is able to catalyze uracil reduction using reduced Fdx as the electron source.Here we report the biochemical characterization of recombinant Pyd Ac.To investigate this hypothesis,we first compared the domain structure of Pyd Ac with that of the previously studied Pyd A homologs from S.scrofa and Brevibacillus agri through bioinformatics analysis.The result showed that it contains the FMN domain and two Fdx domains in a single ORF,but lacked the FAD and NADPH domains.A variety of experiments were also performed.C.chromiireducens Pyd Ac was recombinantly produced in E.coli,and purified to near homogeneity,then followed reconstitution of purified Pyd Ac in vitro.Colorimetric activity assays combined with high-performance liquid chromatography-mass spectrometry(LC-MS)were carried out to detect the production of dihydrouracil and thereby confirmed the activity of Pyd Ac.Pyd Ac can rely on either methyl viologen or Fdx to obtain reducing power to catalyze uracil reduction.C.chromiireducens could grow in defined medium that clarified uracil could be used as the sole nitrogen source for bacterial growth.In addition,it was discovered through bioinformatics that the majority of Pyd Ac sequences belong to strict anaerobic bacteria in the order Clostridiales,including C.chromiireducens Pyd Ac.These homologs likely do not use NAD(P)H as a reductant and may use reduced Fdx as a reductant,consistent with the physiological characteristics of these bacteria.Moreover,the findings not only have vital theoretical significance for studying the diversity of microbial metabolism and synthetic biology but have potential application value.