Purification, crystallization and preliminary X-ray analysis of native and selenomethionine class I tagatose-1,6-bisphosphate aldolase fromStreptococcus pyogenes

Abstract

Tagatose-1,6-bisphosphate aldolase (EC 4.1.2.40) is situated at the branching of the tagatose-6-phosphate and Embden-Meyerhof-Parnas (glycolysis) metabolic pathways, where it catalyzes the reversible cleavage of tagatose-1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. The recombinant protein from Streptococcus pyogenes was overexpressed in Escherichia coli in its native and selenomethionine-derivative forms and purified using ion-exchange and hydrophobic interaction chromatography. Orthorhombic crystals suitable for structural analysis were obtained by the hanging-drop vapour-diffusion method for both isoforms. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 63.7, b = 108.1, c = 238.7 A for the native form and a = 64.1, b = 108.3, c = 239.8 A for the selenomethionine derivative. The asymmetric unit contains four protomers, corresponding to a crystal volume per protein weight (V(M)) of 2.8 A(3) Da(-1) and a solvent content of 56% by volume.

Structure of a Class I Tagatose-1,6-bisphosphate Aldolase

“…A control experiment was performed to confirm this result; DHAP was first labeled using TBP aldolase, and the labeled DHAP was then detritiated again using TBP aldolase. 28 and Leu 275 in the TBP aldolase structure, respectively. B, superposition of the crystal structures of native (pale cyan) and DHAP-bound (cyan) TBP aldolase with native (pale magenta) and DHAP-bound (magenta) FBP aldolase.…”

“…Together, these considerations explain the inability to detect pro-R labeling of the DHAP C3 hydrogen in FBP aldolase (16). The loss of stereospecific labeling at C3 in TBP aldolase is thus a likely consequence of cis-trans isomerization of the carbanion promoted by Gln 28 and Leu 275 . From an evolutionary perspective, C3 epimerization in class I DHAP-dependent aldolases would be based on a conserved stereospecific proton transfer mechanism exploiting cis-trans isomerization in the carbanion intermediate.…”

“…The DHAP C3 hydroxyl, by adopting a trans configuration in Fig. 4, forms a hydrogen bond with DHAP O1 (2.4 -2.6 Å) that would be further stabilized by hydrogen bonding with Gln 28 . The observed 2:1 ratio in the rates of pro-S and pro-R labeling at C3 of DHAP in TBP aldolase is consistent with a mechanism of stereofacial si face proton transfer at C3 in both E and Z isomers with more extensive hydrogen bonding stabilizing the E isomer.…”

“…Recombinant TBP aldolase was purified to homogeneity using a three-step chromatographic protocol as reported previously (28). Briefly, cells were lysed at 4°C, and the lysate was applied onto an anion exchange column (DEAE-Sepharose Fast Flow) and eluted with a NaCl gradient at pH 7.5.…”

“…MAD Phasing and Refinement-The three-dimensional structure of TBP aldolase from S. pyogenes was solved using MAD phasing on previously collected data of a SeMet crystal (28). The three different wavelength (inflection, peak, and remote) MAD data sets allowed the program SOLVE (31) to determine the positions of five selenium atoms of six possible in each protomer and to estimate initial protein phases.…”