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

Abstract

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...

Crystal Structure and Substrate Specificity of D-Galactose-6-Phosphate Isomerase Complexed with Substrates

“…The three-dimensional structure and mechanism of action of tagatose-6-phosphate kinase (LacC) [14] and of class I and II tagatose-1,6-diphosphate aldolase (LacD) [15,16] have been investigated using X-ray crystallography techniques. Galactose-6-phosphate isomerase (LacAB), which is a heteromultimer of LacA and LacB subunits, has been cloned, expressed in E.…”

Broadening Deoxysugar Glycodiversity: Natural and Engineered Transaldolases Unlock a Complementary Substrate Space

“…This correlates with numerous observations using FruA and other DHAP-dependent aldolases, for which hydroxylation at the 2-or 3-position facilitates the correct binding of the aldehyde electrophile in the active site. [16,31] It became evident from X-ray studies of DHAP aldolases that the absolute stereospecificity of the configuration at the nucleophilic carbon usually is fully controlled by the enzyme as stereospecific deprotonation is assisted by hydrogen bonding of the hydroxy group to give a cis or trans enediolate (or hydroxyenamine equivalent) with spatial protection of one prochiral hemisphere of the "carbanion" equivalent from the protein backbone. Diastereoselectivity results from two-fold hydrogen bonding of the aldehyde carbonyl by active-site residues to activate and correctly position the electrophilic center for stereoselective attack by the enolate (or enamine) nucleophile with retention of its configuration.…”

Enzyme‐Catalyzed Aldol Additions

“…Interestingly, it has been observed that tagatose aldolase from Staphylococcus aureus is able to catalyze the aldol addition of DHAP to d-glyceraldehyde-3-phosphate to produce a mixture of the 1,6-diphosphate derivatives of d-tagatose, d-fructose, d-sorbose, and d-psicose [96]. A similar stereoselective behavior has been observed in TagA from Streptococcus pyogenes [97]. Investigations on the structure and mechanism of this aldolase revealed that its lack of chiral discrimination with respect to the configuration of hydroxyl groups at C-3 position, the most conserved in the aldolases known, is given by the ability of the enzyme to interchange the cis (Scheme 8.27a) and trans (Scheme 8.27b) isomers with respect to the DHAP C2-Lys205 N ε bond of the intermediate enzyme-DHAP carbanion mesomer (Scheme 8.27).…”