Tagatose-1,6-bisphosphate 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-bisphosphate, sorbose 1,6-bisphosphate, and tagatose 1,6-bisphosphate) to dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde 3-phosphate 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 (␣/␤) 8 fold in each dimeric subunit. Flash-cooled crystals of native enzyme soaked with dihydroxyacetone phosphate trapped a covalent intermediate with carbanionic character at Lys 205 , 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-labeled DHAP at C3 hydrogen were carried out in both solution and crystalline state to assess stereochemical control at C3. The kinetic studies show labeling at both pro-R and pro-S C3 positions of DHAP yet detritiation only at the C3 pro-S-labeled 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 tagatose-1,6-bisphosphate aldolase.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 (2-4). Tagatose-1,6-bisphosphate (TBP) 2 aldolase is an inducible enzyme that, although demonstrating greatest affinity for D-tagatose 1,6-bisphosphate, can also use as substrate the bisphosphorylated D-hexose stereoisomers: sorbose bisphosphate, psicose bisphosphate, and fructose bisphosphate (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 and dihydroxyacetone phosphate (DHAP), whereas the condensation of glyceraldehyde 3-phosphate 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 covalent Schiff base intermediates (6 -8), whereas class I...

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