Structure of a Class I Tagatose-1,6-biphosphate Aldolase :

Tagatose-1,6-biphosphate (TBP) aldolase from Streptococcus pyogenes is a class I aldolase that exhibits a remarkable lack of chiral discrimination with respect to the configuration of hydroxyl groups at both C3 and C4 positions. The enzyme catalyzes the reversible cleavage of four diastereoisomers: fructose-1,6-bisphosphate (FBP), psicose-1,6-bis-phosphate, sorbose-1,6bisphosphate and tagatose-1,6-bisphosphate to dihydroxyacetone-P and D-glyceraldehyde 3-P with high catalytic efficiency. To investigate its enzymatic mechanism, high resolution crystal structures were determined of both native enzyme and native enzyme in complex with dihydroxyacetone-P. The electron density map revealed a (α/β)8 fold in each dimeric subunit. Flash cooled crystals of native enzyme soaked with dihydroxyacetone-P trapped a covalent intermediate with carbanionic character at Lys, different from the enamine mesomer bound in stereospecific class I FBP aldolase. Structural analysis indicates extensive active site conservation with respect to class I FBP aldolases including conserved conformational responses to DHAP binding and conserved stereospecific proton transfer at the DHAP C3 carbon mediated by a proximal water molecule. Exchange reactions with tritiated water and tritium labelled DHAP at C3 hydrogen were carried out in both solution and crystalline state to assess stereochemical control at C3. The kinetic studies show labelling at both pro-R and pro-S C3 positions of DHAP yet detriation only at the C3 pro-S labelled position. Detritiation of the C3 pro-R label was not detected and is consistent with preferential cis-trans isomerism about the C2-C3 bond in the carbanion as the mechanism responsible for C3 epimerization in TBP aldolase. Introduction Aldolases are crucial enzymes in living organisms because of their role in essential metabolic pathways such as gluconeogenesis and glycolysis. Their ability to control the stereochemistry of the carbon-carbon bond formation makes them models for de novo preparation of carbohydrates (1), and ideal alternatives to traditional methods in synthetic organic chemistry (2, 3, 4). Tagatose-1,6bisphosphate (TBP) aldolase is an inducible enzyme which, although demonstrating greatest affinity for D-tagatose-1,6-bisphosphate, can also use as substrate the bisphosphorylated D-hexose stereoisomers: sorbose-P2, psicose-P2 and fructose-P2 (5). The four sugars are diastereoisomers and differ in stereochemistry at carbon 3 and at carbon 4 with respect to the configuration of their hydroxyl groups. The cleavage of the four sugars produces glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP), while the condensation of G3P and DHAP produces a mixture of the four D-hexoses in Staphylococcus aureus (5). Aldolases are broadly categorized with respect to their catalytic mechanism into two classes. Class I aldolases are characterized by formation of a covalent Schiff base intermediates (6, 7, 8), while class II aldolases are metallo-dependant enzymes and use a divalent transition metal ion to polarize the substrate ketose (9, 10). Of all aldolases, class I FBP aldolase from rabbit muscle has been the most extensively studied (11, 12). To form the FBP C3C4 bond in aldol condensation, the enzyme stereospecifically abstracts the pro-S C3 proton of the iminium intermediate formed by a lysine residue in the active site with DHAP (13, 14, 15), thereby generating the carbanionic character at C3 of DHAP for the aldol reaction. The nascent carbon-carbon bond has the same orientation as the pro-S αhydrogen initially abstracted from the DHAP imine http://www.jbc.org/cgi/doi/10.1074/jbc.M109.080358 The latest version is at JBC Papers in Press. Published on May 1, 2010 as Manuscript M109.080358