KLINKE Sebastian
congresos y reuniones científicas
Rhizobiales bear an atypical riboflavin pathway. (Poster)
Newport, Rhode Island, EEUU
Congreso; Gordon Research Conference "Microbial adhesion and signal transduction"; 2007
Institución organizadora:
Gordon Research Conferences
Riboflavin is the precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), essential cofactors for a multitude of mainstream metabolic enzymes that mediate hydride, oxygen, and electron transfer reactions. Only plants, fungi and microorganisms can synthesize riboflavin, whereas higher animals, including man, must obtain it through diet. The last two steps in the biosynthesis of riboflavin are catalyzed by 6,7-dimethyl-8-ribityllumazine synthase (LS) and riboflavin synthase. LS is known to exhibit different quaternary assemblies between species, existing as free pentamers, decamers (dimers of pentamers) and icosahedrally arranged dodecamers of pentamers. A phylogenetic analysis on eubacterial, fungal and plant LSs allowed us to classify them into two categories: Type-I LSs (pentameric or icosahedral) and Type-II LSs (decameric). The Rhizobiales represent an order of á-proteobacteria that includes, among others, the genera Mesorhizobium, Agrobacterium and Brucella. Here, we present structural and kinetic studies on several LSs from Rhizobiales. Interestingly, Mesorhizobium and Brucella encode both a Type-I and a Type-II LS called RibH1 and RibH2 respectively. We show that Type-II LSs appear to be almost inactive, whereas Type-I LSs present a highly variable catalytic activity according to the genus. Additionally, we have solved four RibH1 / RibH2 crystallographic structures from the genera Mesorhizobium and Brucella. The relationship between the active site architecture and catalytic properties in these isoenzymes is discussed, and a model that describes the enzymatic behavior is proposed. Furthermore, sequence alignment studies allowed us to extend our results to the genus Agrobacterium. Our results suggest that the selective pressure controlling the riboflavin pathway favored the evolution of catalysts with low reaction rates, since the excess of flavins in the intracellular pool in Rhizobiales could act as a negative factor when these bacteria are exposed to oxidative or nitrosative stress. RibH2 is potentially regulated by an RFN-box, a highly conserved RNA-regulatory element, found frequently in untranslated regions of prokaryotic mRNAs. The RFN element has an important role in the regulation of both transcription and translation of riboflavin biosynthesis genes. The proposed mechanism of regulation for ribH2 gene in Brucella is an attenuation of translation by sequestering of the Shine-Dalgarno box in response to FMN. We have produced null mutants of B. abortus in RibH1 and RibH2. The absence of RibH1 does not affect the virulence of the bacteria, whereas the absence of RibH2 produces a marked decrease in the virulence. This effect is observed ex vivo, since the mutation affects the replication of B. abortus in a murine macrophagic cell line. The effect is also observed so in vivo, since the mutation dramatically affects the persistence of B. abortus in spleens of infected mice. The lack of RibH2 also increases the susceptibility of the bacteria to oxidative stress produced by hydrogen peroxide. All these results show that RibH2 is a virulence factor, which confers resistance to oxidative stress.