Crystal structures of the metal-dependent 2-dehydro-3-deoxy-galactarate aldolase suggest a novel reaction mechanism

Abstract

Carbon-carbon bond formation is an essential reaction in organic chemistry and the use of aldolase enzymes for the stereochemical control of such reactions is an attractive alternative to conventional chemical methods. Here we describe the crystal structures of a novel class II enzyme, 2-dehydro-3-deoxy-galactarate (DDG) aldolase from Escherichia coli, in the presence and absence of substrate. The crystal structure was determined by locating only four Se sites to obtain phases for 506 protein residues. The protomer displays a modified (alpha/beta)(8) barrel fold, in which the eighth alpha-helix points away from the beta-barrel instead of packing against it. Analysis of the DDG aldolase crystal structures suggests a novel aldolase mechanism in which a phosphate anion accepts the proton from the methyl group of pyruvate.

Crystal structure of a bifunctional aldolase–dehydrogenase: Sequestering a reactive and volatile intermediate

“…Aldolase enzymes fall into two classes: those that rely on an active site lysine residue to assist in catalysis (class 1), and those in which aldol cleavage is assisted by divalent metal ions (class II). Structures are available for four class II aldolases: L-fuculose-1-phosphate aldolase (34,35), fructose-1,6-bisphosphate aldolase (36, 37), tagatose-1,6-bisphosphate aldolase (38), and 2-dehydro-3-deoxygalactarate aldolase (18). The three latter enzymes display an (␣͞␤) 8 fold, whereas fuculose-1-phosphate aldolase does not.…”

“…The experimental phases were extended to 1.7-Å resolution by using the free atom refinement method in ARP͞WARP (18), and automatic chain tracing was carried out. The model was subjected to refinement using the program REFMAC (19).…”

The role of a conserved histidine residue in a pyruvate‐specific Class II aldolase

“…Crystal structures of DDG aldolase [6] and HpcH/HpaI [7] show common (ab) 8 TIM barrel folds with similar active site architectures. Pyruvate or the pyruvate analogue, oxamate, binds in a bidentate fashion, via the C1 carboxylate and C2 carbonyl, to the divalent metal ion cofactor.…”

“…It is not apparent from structural data which residues could participate in this acid/base catalysis since there are no candidate residues close enough to C3 of pyruvate, and a structure of the enzymes in complex with the aldehyde moiety (containing the C4 carbon of the condensed product) is unavailable. An enzyme bound phosphate was previously postulated to be the catalytic acid [6], but this was subsequently disputed since in phosphate-free buffer, pyruvate proton exchange catalyzed by HpaI is not impaired [2]. A histidine residue (His 45) within the active site of HpcH has been replaced with alanine and the resulting enzyme was reported to be inactive [7].…”

Crystal Structure of Reaction Intermediates in Pyruvate Class II Aldolase

“…The enzyme adopts a triose-phosphate isomerase barrel fold with helix 8 of the (␣␤) 8 barrel in each subunit, forming a domain-swapped dimer with a neighboring subunit in an overall hexameric structure. This structure resembles that of its homologs, 2-dehydro-3-deoxygalactarate aldolase (8) and 2-dehydro-3-deoxy-L-rhamnonate aldolase (YfaU) (16), which are involved in sugar metabolism in microorganisms.…”