RESUMO
The "extended" type of short chain dehydrogenases/reductases (SDR), share a remarkable similarity in their tertiary structures inspite of being highly divergent in their functions and sequences. We have carried out principal component analysis (PCA) on structurally equivalent residue positions of 10 SDR families using information theoretic measures like Jensen-Shannon divergence and average shannon entropy as variables. The results classify residue positions in the SDR fold into six groups, one of which is characterized by low Shannon entropies but high Jensen-Shannon divergence against the reference family SDR1E, suggesting that these positions are responsible for the specific functional identities of individual SDR families, distinguishing them from the reference family SDR1E. Site directed mutagenesis of three residues from this group in the enzyme UDP-Galactose 4-epimerase belonging to SDR1E shows that the mutants promote the formation of NADH containing abortive complexes. Finally, molecular dynamics simulations have been used to suggest a mechanism by which the mutants interfere with the re-oxidation of NADH leading to the formation of abortive complexes.
Assuntos
Proteínas de Escherichia coli/química , Modelos Moleculares , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Oxirredutases/química , UDPglucose 4-Epimerase/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Biocatálise , Domínio Catalítico , Sequência Conservada , Bases de Dados de Proteínas , Entropia , Proteínas de Escherichia coli/classificação , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/classificação , Isoenzimas/genética , Isoenzimas/metabolismo , Simulação de Dinâmica Molecular , Análise Multivariada , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Oxirredutases/classificação , Oxirredutases/genética , Oxirredutases/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/classificação , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Análise de Componente Principal , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência , Terminologia como Assunto , UDPglucose 4-Epimerase/classificação , UDPglucose 4-Epimerase/genética , UDPglucose 4-Epimerase/metabolismoRESUMO
A clone that complements mutations in Yersinia enterocolitica lipopolysaccharide (LPS) core biosynthesis was isolated, and the DNA sequence of the clone was determined. Three complete open reading frames and one partial open reading frame were located on the cloned DNA fragment. The first, partial, open reading frame had homology to the rfbK gene. The remaining reading frames had homology to galE, rol, and gsk. Analysis of the galE homolog indicates that although it can complement an Escherichia coli galE mutant, its primary function in Y. enterocolitica is not in the production of UDP galactose but, instead, some other nucleotide sugar required for LPS biosynthesis. This gene has been renamed lse, for LPS sugar epimerase. The rol homolog has been demonstrated to have a role in Y. enterocolitica serotype 0:8 O-polysaccharide antigen chain length determination. An additional galE homolog has been identified in Y. enterocolitica by homology to the E. coli gene. The product of this gene has UDP galactose 4-epimerase activity in both E. coli and Y. enterocolitica. This gene is linked to the other genes of the galactose utilization pathway, similar to what is seen in other members of the family Enterobacteriaceae. Although Y. enterocolitica 0:8 strains are reported to have galactose as a constituent of LPS, a strain containing a mutation in this galE gene does not exhibit any LPS defects.
