The Crystallographic And Functional Research On Tal2 From Saccharomyces Cerevisiae And DDG Aldolase From Leptospira Interrogans

Aldolases form a big family of cleavage enzymes.The aldolase sensu stricto means 1,6-diphosphate-D-fructose aldolase(EC 4.1.2.13).It can reversible catalyse the cleavage of 1,6-diphosphate-D-fructose,forming 3-phosphate-D-glyceraldehyde andα-phosphate-dihydroxyacetone.The generalized aldolase contains all enzymes which catalyzes the similar reaction.Transaldolase(EC 2.2.1.2) and 2-dehydro-3-deoxyglucarate aldolase mentioned in this paper belong this aldolase family.Aldolase catalyse carbon-carbon bond formation and cleavage of metabolic substance in glycolysis.Aldol condensation is one of the most important reactions in synthetic organic chemistry and aldolase is therefore of considerable interest as a biocatalyst.Based on their reaction mechanism,aldolases have been classified into two families,classâ… and classâ…¡.The classâ… enzymes are generally found in higher organisms and classâ…¡enzymes are found in bacteria and other lower organisms.The classâ… aldolases catalyze chemical reactions using a Schiff base mechanism to stabilize reaction intermediates.A common feature in classâ… aldolases is a lysine residue which forms the Schiff base with the substrate in the active site,whereas in the classâ…¡aldolases is a divalent metal ion for intermediate stabilization.Transaldolase,one member of classâ… aldolases,catalyzes the reversible transfer of a dihydroxyacetone moiety from 6-phosphate-fructose to 4-phosphate-erythrose, giving 7-phosphate-sedoheptulose and 3-phosphate-glyceraldehyde in the nonoxidative branch of the pentose phosphate pathway.One open reading frame in Saccharomyces cerevisiae genome,named NQM1/YGR043C,exhibits more than 50%sequence identity to the family of transaldolases,was proposed to be a putative transaldolase in yeast.However,no experimental evidence has been reported to prove its transaldolase activity to date.2-dehydro-3-deoxygalactarate aldolase from Leptospira interrogans,one member of the classâ…¡aldolase family,catalyzes the reversible aldol cleavage of 2-dehydro-3-deoxygalactarate to pyruvate and tartronic semialdehyde in the pyruvate-metabolism pathway.It attracts many health scientists' attentions as a potential novel antibiotic target.(â… ) We cloned the NQM1/YGR043C gene from the yeast genome into a pET22b(+) vector and expressed the protein in Escherichia coli strain BL21(DE3). Also we used the hanging-drop vapor diffusion method and obtained the crystal.The crystal belongs to the space group I2_â… 2_â… 2_â… ,with unit-cell parameters a=85.03(?), b=113.46(?),c=158.92(?),α=β=γ=90°.The crystal structure was determined by the molecular replacement method and refined to a 1.9(?) resolution.The NQM1/YGR043C protein folds into an eight-stranded(α/β)₈ barrel,Sevenα-helices(with absence ofα1 helix) embraced theβ-barrel.The core structure was further surrounded by seven additional helices(αA-αG helices),which is conserved among all reported transaldolase structures.Three dimensional structure comparisons revealed that both the overall structure and the core(α/β)₈ barrel of NQM1/YGR043C are similar to those in other transaldolases.Alignments of the sequence and structure of NQM1/YGR043C with other transaldolases indicated that this protein contained all residues carrying out the transaldolase activity in the catalytic sites that had been reported and these residues were located at the same spatial positions within the active sites identified in other transaldolases.The enzyme activity assays further demonstrated its transaldolase activity.Based on these data,we concluded that the open reading frame of NQM1/YGR043C does encode a transaldolase,which could be named as TAL2_YEAST.(â…¡) We cloned the 2-dehydro-3-deoxygalactarate aldolase gene of Leptospira interrogans into a pET22b(+) vector,expressed the protein in Escherichia coli strain BL21(DE3) and purified it using a DEAE-Sepharose column and a Superdex 75 size exclusion chromatography column.Also we used the hanging-drop vapor diffusion method and obtained the crystal.The crystal's diffraction ability was strengthened by optimizing the cytoprotection.The crystal belongs to the space group C2,with unit-cell parameters a=293.5(?),b=125.6(?),c=87.6(?),α=γ=90°,β=100.9°.Also we obtained the primary structure model by the molecular replacement method.