RESUMO
Phosphomannomutase (PMM) catalyses the conversion of mannose-6-phosphate to mannose-1-phosphate, an essential step in mannose activation and the biosynthesis of glycoconjugates in all eukaryotes. Deletion of PMM from Leishmania mexicana results in loss of virulence, suggesting that PMM is a promising drug target for the development of anti-leishmanial inhibitors. We report the crystallization and structure determination to 2.1 A of L. mexicana PMM alone and in complex with glucose-1,6-bisphosphate to 2.9 A. PMM is a member of the haloacid dehalogenase (HAD) family, but has a novel dimeric structure and a distinct cap domain of unique topology. Although the structure is novel within the HAD family, the leishmanial enzyme shows a high degree of similarity with its human isoforms. We have generated L. major PMM knockouts, which are avirulent. We expressed the human pmm2 gene in the Leishmania PMM knockout, but despite the similarity between Leishmania and human PMM, expression of the human gene did not restore virulence. Similarities in the structure of the parasite enzyme and its human isoforms suggest that the development of parasite-selective inhibitors will not be an easy task.
Assuntos
Leishmania mexicana/enzimologia , Fosfotransferases (Fosfomutases)/química , Animais , Cristalografia por Raios X , Humanos , Isoenzimas/química , Isoenzimas/genética , Leishmania mexicana/genética , Leishmania mexicana/patogenicidade , Fosfotransferases (Fosfomutases)/genética , Fosfotransferases (Fosfomutases)/isolamento & purificação , Homologia Estrutural de Proteína , Virulência/genéticaRESUMO
Azotobacter vinelandii is a soil gamma-proteobacteria that fixes nitrogen and forms desiccation-resistant cysts. The exopolysaccharide alginate is an integral part of the layers surrounding the cysts. Here, we reported the cloning of A. vinelandii algC, encoding the enzyme catalyzing the second step of alginate pathway. We showed that AlgC is involved not only in alginate production, but also in lipopolysaccharide (LPS) synthesis and that it seems to have both phosphomannomutase and phosphoglucomutase activities. The transcriptional analysis of the A. vinelandii algC gene showed that it contained two start sites, one of which was dependent on the alternative sigma factor AlgU/AlgT. This finding explains why alginate biosynthesis is dependent on AlgU activity, since all other alginate biosynthetic genes have been characterized previously and algC is the only alginate structural gene that is directly transcribed by this sigma factor.