A Pathway For Degradation Of Uracil To Acetyl Coenzyme A In Bacillus Megaterium

Bacteria utilize diverse biochemical pathways for the degradation of the pyrimidine ring.The function of the pathways studied to date has been the release of nitrogen for assimilation.Three pyrimidine degradation pathways have been reported in bacteria,known as the reductive(Pyd),oxidative,and py Rimidine UTilization(RUT)pathways.The most widespread of these pathways is the reductive pyrimidine catabolic pathway,which converts uracil into ammonia,carbon dioxide andβ-alanine.Recently,we reported an extended Pyd pathway in Lysinibacillus massiliensis,involving aβ-alanine:2-oxoglutarate aminotransferase(Pyd D1),and a NAD(P)H-dependent malonic semialdehyde reductase(Pyd E).These two enzymes convertβ-alanine into 3-hydroxypropionate(3-HP),releasing the nitrogen that remains inβ-alanine.Our study extends the previously reported reduction pathway,enabling the assimilation of the pyrimidine nitrogen atom.Detailed biochemical pathways have been investigated with focus mainly on nitrogen assimilation in the past decades,with only few reports on bacterial carbon metabolism.Here we discover and characterize aβ-alanine:pyruvate aminotransferase(Pyd D2)and a NAD~+-dependent malonic semialdehyde dehydrogenase(MSDH),both from a Pyd gene cluster in Bacillus megaterium.These two enzymes catalyze the conversion ofβ-alanine to acetyl-Co A.The last step catalyzed by MSDH is an oxidative reaction.Unlike the previously characterized L.massiliensis Pyd D1,which exhibits a preference for 2-oxoglutarate as the amine acceptor,B.megaterium Pyd D2 is specific for pyruvate as the amine acceptor.We further demonstrate that B.megaterium can grow in a defined medium with uracil as its sole carbon and energy source,consistent with the physiological relevance of this extended reduction pathway.Our bioinformatics studies also reveal that homologs of Pyd D2 and MSDH are prevalent in many Gram-positive bacteria in the order Bacillales,in association with reductive pyrimidine pathway genes.Taken together,we report our findings of two new enzymes,Pyd D2 and MSDH,involved in a new type of pyrimidine degradation,providing bacteria carbon and energy source.With these findings we expand our understanding of the biodiversity of pyrimidine metabolism and bacterial physiology and the new enzymes could become useful tools in synthetic biology that may have potential industrial applications.