Interrogating l-fuconate dehydratase with tartronate and 3-hydroxypyruvate reveals subtle differences within the mandelate racemase-subgroup of the enolase superfamily.

Enzymes of the enolase superfamily share a conserved structure and a common partial reaction (i.e., metal-assisted, Brønsted base-catalyzed enol(ate) formation). The architectures of the enolization apparatus at the active sites of the mandelate racemase (MR)-subgroup members MR and l-fuconate dehydratase (FucD) are almost indistinguishable at the structural level. Tartronate and 3-hydroxypyruvate (3-HP) recognize the enolization apparatus and can be used to interrogate the active sites for differences that may not be apparent from structural data. We report a circular dichroism-based assay of FucD activity that monitors the change in ellipticity at 216 nm (Δ[Θ]S-P = 8985 ± 87 deg cm2 mol-1) accompanying the conversion of l-fuconate to 2-keto-3-deoxy-l-fuconate. Tartronate was a linear mixed-type inhibitor of FucD (Ki = 8.4 ± 0.7 mM, αKi = 63 ± 11 mM), binding 18-fold weaker than l-fuconate, compared with 2-fold weaker binding of tartronate by MR relative to mandelate. 3-HP irreversibly inactivated FucD (kinact/KI = 0.018 ± 0.002 M-1s-1) with an efficiency that was ∼4.6 × 103-fold less than that observed with MR. The inactivation arose predominantly from modifications at multiple sites and Tris-HCl, but not l-fuconate, afforded protection against inactivation. Similar to the reaction of 3-HP with MR, 3-HP modified the Brønsted base catalyst (Lys 220) at the active site of FucD, which was facilitated by the Brønsted acid catalyst His 351. Thus, the interactions of tartronate and 3-HP with MR and FucD revealed differences in binding affinity and reactivity that differentiated between the enzymes' enolization apparatuses.

Metabolomics of Dietary Acid Load and Incident Chronic Kidney Disease.

OBJECTIVE: Blood biomarkers of dietary intake are more objective than self-reported dietary intake. Metabolites associated with dietary acid load were previously identified in 2 chronic kidney disease (CKD) populations. We aimed to extend these findings to a general population, replicating their association with dietary acid load, and investigating whether the individual biomarkers were prospectively associated with incident CKD. METHODS: Among 15,792 participants in the Atherosclerosis Risk in Communities cohort followed up from 1987 to 1989 (baseline) to 2019, we evaluated 3,844 black and white men and women with dietary and metabolomic data in cross-sectional and prospective analyses. We hypothesized that a higher dietary acid load (using equations for potential renal acid load and net endogenous acid production) was associated with lower serum levels of 12 previously identified metabolites: indolepropionylglycine, indolepropionate, N-methylproline, N-δ-acetylornithine, threonate, oxalate, chiro-inositol, methyl glucopyranoside, stachydrine, catechol sulfate, hippurate, and tartronate. In addition, we hypothesized that lower serum levels of these 12 metabolites were associated with higher risk of incident CKD. RESULTS: Eleven out of 12 metabolites were significantly inversely associated with dietary acid load, after adjusting for demographics, socioeconomic status, health behaviors, health status, and estimated glomerular filtration rate: indolepropionylglycine, indolepropionate, N-methylproline, threonate, oxalate, chiro-inositol, catechol sulfate, hippurate, methyl glucopyranoside (α + ß), stachydrine, and tartronate. N-methylproline was inversely associated with incident CKD (hazard ratio: 0.95, 95% confidence interval: 0.91, 0.99, P = .01). The metabolomic biomarkers of dietary acid load significantly improved prediction of elevated dietary acid load estimated using dietary data, beyond covariates (difference in C statistics: 0.021-0.077, P ≤ 1.08 × 10-3). CONCLUSION: Inverse associations between candidate biomarkers of dietary acid load were replicated in a general population. N-methylproline, representative of citrus fruit consumption, is a promising marker of dietary acid load and could represent an important pathway between dietary acid load and CKD.

Tartronic acid promotes de novo lipogenesis and inhibits CPT-1ß by upregulating acetyl-CoA and malonyl-CoA.

As a dicarboxylic acid with the structural formula HOOCCH (OH) COOH, tartronic acid is considered as an inhibitor of the transformation of carbohydrates into fat under fat-deficient diet conditions. However, the effect of tartronic acid on lipogenesis under high-fat diet conditions has yet to be established. In this work, we investigated the regulatory role of tartronic acid in lipogenesis in 3T3-L1 adipocytes and C57BL/6J mice. The results confirmed that tartronic acid promoted weight gain (without affecting food intake) and induced adipocyte hypertrophy in epididymal white adipose tissue and lipid accumulation in the livers of high-fat diet-induced obese mice. In vitro, tartronic acid promoted 3T3-L1 adipocyte differentiation by increasing the protein expression of FABP-4, PPARγ and SREBP-1. Moreover, the contents of both acetyl-CoA and malonyl-CoA were significantly upregulated by treatment with tartronic acid, while the protein expression of CPT-1ß were inhibited. In summary, we proved that tartronic acid promotes lipogenesis by serving as substrates for fatty acid synthesis and inhibiting CPT-1ß, providing a new perspective for the study of tartronic acid.

Separation of tartronic and glyceric acids by simulated moving bed chromatography.

The SMB unit developed by the Laboratory of Separation and Reaction Engineering (FlexSMB-LSRE®) was used to perform tartronic acid (TTA) and glyceric acid (GCA) separation and to validate the mathematical model in order to determine the optimum operating parameters of an industrial unit. The purity of the raffinate and extract streams in the experiments performed were 80% and 100%, respectively. The TTA and GCA productivities were 79 and 115 kg per liter of adsorbent per day, respectively and only 0.50 cubic meters of desorbent were required per kilogram of products. Under the optimum operating conditions, which were determined through an extensive simulation study based on the mathematical model developed to predict the performance of a real SMB unit, it was possible to achieve a productivity of 86 kg of TTA and 176 kg of GCA per cubic meter of adsorbent per day (considering the typical commercial purity value of 97% for both compounds) with an eluent consumption of 0.30 cubic meters per kilogram of products.

Participation of phosphofructokinase, malate dehydrogenase and isocitrate dehydrogenase in capacitation and acrosome reaction of boar spermatozoa.

The aim of this work was to determine the enzymatic activity of phosphofructokinase (PFK), malate dehydrogenase (MDH) and isocitrate dehydrogenase (IDH) in boar spermatozoa and study their participation in bicarbonate-induced capacitation and follicular fluid-induced acrosome reaction. Enzymatic activity of these enzymes was determined spectrophotometrically in extracts of boar spermatozoa. Sperm suspensions were incubated in the presence of bicarbonate (40 mM), a well-known capacitation inducer, or follicular fluid (30%), as an acrosome reaction inducer, and different concentrations of oxoglutarate, oxalomalate and hydroxymalonate, inhibitors of PFK, IDH and MDH, respectively. Capacitation percentages were determined by the fluorescence technique of chlortetracycline (CTC), and true acrosome reaction was determined by trypan blue and differential-interferential contrast, optical microscopy. The activity of PFK in boar spermatozoa enzymatic extracts was 1.70 ± 0.19 U/1010 spermatozoa, the activity of NAD- and NADP-dependent IDH was 0.111 ± 0.005 U/1010 and 2.22 ± 0.14 U/1010 spermatozoa, respectively, and the activity of MDH was 4.24 ± 0.38 U/1010 spermatozoa. The addition of the specific inhibitors of these enzymes prevented sperm capacitation and decreased sperm motility during capacitation and inhibited the acrosome reaction (AR), without affecting the sperm motility during this process. Our results demonstrate the participation of PFK, IDH and MDH in bicarbonate-induced capacitation and follicular fluid-induced acrosome reaction in boar spermatozoa, contributing to elucidate the mechanisms that produce energy necessary for these processes in porcine spermatozoa.

Protective Effects of Tinospora cordifolia on Hepatic and Gastrointestinal Toxicity Induced by Chronic and Moderate Alcoholism.

AIMS: Heavy alcohol intake depletes the plasma vitamins due to hepatotoxicity and decreased intestinal absorption. However, moderate alcohol intake is often thought to be healthy. Therefore, effects of chronic moderate alcohol intake on liver and intestine were studied using urinary vitamin levels. Furthermore, effects of Tinospora cordifolia water extract (TCE) (hepatoprotective) on vitamin excretion and intestinal absorption were also studied. METHODS: In the study, asymptomatic moderate alcoholics (n = 12) without chronic liver disease and healthy volunteers (n = 14) of mean age 39 ± 2.2 (mean ± SD) were selected and divided into three groups. TCE treatment was performed for 14 days. The blood and urine samples were collected on Day 0 and 14 after treatment with TCE and analyzed. RESULTS: In alcoholics samples, a significant increase in the levels of gamma-glutamyl transferase, aspartate transaminase, alanine transaminase, Triglyceride, Cholesterol, HDL and LDL (P < 0.05) was observed but their level get downregulated after TCE intervention. Multivariate analysis of metabolites without missing values showed an increased excretion of 7-dehydrocholesterol, orotic acid, pyridoxine, lipoamide and niacin and TCE intervention depleted their levels (P < 0.05). In contrast, excretion of biotin, xanthine, vitamin D2 and 2-O-p-coumaroyltartronic acid (CA, an internal marker of intestinal absorption) were observed to be decreased in alcoholic samples; however, TCE intervention restored the CA and biotin levels. Vitamin metabolism biomarkers, i.e. homocysteine and xanthurenic acid, were also normalized after TCE intervention. CONCLUSION: Overall data depict that moderate alcohol intake is also hepatotoxic and decreases intestinal absorption. However, TCE treatment effectively increased the intestinal absorption and retaining power of liver that regulated alcohol-induced multivitamin deficiency.

A polyketide synthase acyltransferase domain structure suggests a recognition mechanism for its hydroxymalonyl-acyl carrier protein substrate.

We have previously shown that the acyl transferase domain of ZmaA (ZmaA-AT) is involved in the biosynthesis of the aminopolyol polyketide/nonribosomal peptide hybrid molecule zwittermicin A from cereus UW85, and that it specifically recognizes the precursor hydroxymalonyl-acyl carrier protein (ACP) and transfers the hydroxymalonyl extender unit to a downstream second ACP via a transacylated AT domain intermediate. We now present the X-ray crystal structure of ZmaA-AT at a resolution of 1.7 Å. The structure shows a patch of solvent-exposed hydrophobic residues in the area where the AT is proposed to interact with the precursor ACP. We addressed the significance of the AT/ACP interaction in precursor specificity of the AT by testing whether malonyl- or methylmalonyl-ACP can be recognized by ZmaA-AT. We found that the ACP itself biases extender unit selection. Until now, structural information for ATs has been limited to ATs specific for the CoA-linked precursors malonyl-CoA and (2S)-methylmalonyl-CoA. This work contributes to polyketide synthase engineering efforts by expanding our knowledge of AT/substrate interactions with the structure of an AT domain that recognizes an ACP-linked substrate, the rare hydroxymalonate. Our structure suggests a model in which ACP interaction with a hydrophobic motif promotes secondary structure formation at the binding site, and opening of the adjacent substrate pocket lid to allow extender unit binding in the AT active site.

Phosphofructokinase and malate dehydrogenase participate in the in vitro maturation of porcine oocytes.

Oocyte maturation depends on the metabolic activity of cumulus-oocyte complex (COC) that performs nutritive and regulatory functions during this process. In this work, the enzymes [phosphofructokinase (PFK) and malate dehydrogenase (MDH)] were tested to elucidate the metabolic profile of porcine COCs during the in vitro maturation (IVM). Enzymatic activity was expressed in U/COC and U/mg protein (specific activity) as mean ± SEM. In vitro maturation was performed with 2-oxoglutarate (5, 10 and 20 mm) or hydroxymalonate (30, 60 and 100 mm) inhibitors of PFK and MDH, respectively. The PFK and MDH activities (U) remained constant during maturation. For PFK, the U were (2.48 ± 0.23) 10(-5) and (2.54 ± 0.32) 10(-5) , and for MDH, the U were (4.72 ± 0.42) 10(-5) and (4.38 ± 0.25) 10(-5) for immature and in vitro matured COCs, respectively. The specific activities were significantly lower after IVM, for PFK (4.29 ± 0.48) 10(-3) and (0.94 ± 0.12) 10(-3) , and for MDH (9.08 ± 0.93) 10(-3) and (1.89 ± 0.10) 10(-3) for immature and in vitro matured COCs, respectively. In vitro maturation percentages and enzymatic activity diminished with 20 mm 2-oxoglutarate or 60 mm hydroxymalonate (p < 0.05). Viability was not affected by any concentration of the inhibitors evaluated. The U remained unchanged during IVM; however, the increase in the total protein content per COC provoked a decrease in the specific activity of both enzymes. Phosphofructokinase and MDH necessary for oocyte IVM would be already present in the immature oocyte. The presence of inhibitors of these enzymes impairs the meiotic maturation. Therefore, the participation of these enzymes in the energy metabolism of the porcine oocyte during IVM is confirmed in this study.

Photo-redox reactions of dicarboxylates and α-hydroxydicarboxylates at the surface of Fe(III)(hydr)oxides followed with in situ ATR-FTIR spectroscopy.

Colloidal mineral-phases play an important role in the adsorption, transport and transformation of organic and inorganic compounds in the atmosphere and in aqueous environments. Artificial UV-light and sunlight can induce electron transfer reactions between metal ions of the solid phases and adsorbed compounds, leading to their transformation and degradation. To investigate different possible photo-induced oxidation pathways of dicarboxylates adsorbed on iron(III)(hydr)oxide surfaces, we followed UV-A induced photoreactions of oxalate, malonate, succinate and their corresponding α-hydroxy analogues tartronate and malate with in situ ATR-FTIR spectroscopy in immersed particle layers of lepidocrocite, goethite, maghemite and hematite at pH 4. UV-A light (365 ± 5 nm) lead to fast degradation of oxalate, tartronate and malate, while malonate and succinate were photo-degraded at much slower rates. Efficient generation of OH-radicals can be excluded, as this would lead to fast and indiscriminate degradation of all tested compounds. Rapid photo-degradation of adsorbed oxalate and the α-hydroxydicarboxylates must be induced by direct ligand-to-metal charge transfer (LMCT) or by selectively oxidizing valence band holes, both processes requiring inner-sphere coordination with direct ligand-to-metal bonds to enable efficient electron-transfer. The slow photo-degradation of malonate and succinate can be explained by low-yield production of OH-radicals at the surface of the iron(III)(hydr)oxides.

Structures of asymmetric complexes of human neuron specific enolase with resolved substrate and product and an analogous complex with two inhibitors indicate subunit interaction and inhibitor cooperativity.

In the presence of magnesium, enolase catalyzes the dehydration of 2-phospho-d-glycerate (PGA) to phosphoenolpyruvate (PEP) in glycolysis and the reverse reaction in gluconeogensis at comparable rates. The structure of human neuron specific enolase (hNSE) crystals soaked in PGA showed that the enzyme is active in the crystals and produced PEP; conversely soaking in PEP produced PGA. Moreover, the hNSE dimer contains PGA bound in one subunit and PEP or a mixture of PEP and PGA in the other. Crystals soaked in a mixture of competitive inhibitors tartronate semialdehyde phosphate (TSP) and lactic acid phosphate (LAP) showed asymmetry with TSP binding in the same site as PGA and LAP in the PEP site. Kinetic studies showed that the inhibition of NSE by mixtures of TSP and LAP is stronger than predicted for independently acting inhibitors. This indicates that in some cases inhibition of homodimeric enzymes by mixtures of inhibitors ("heteroinhibition") may offer advantages over single inhibitors.

Impairment of aerobic glycolysis by inhibitors of lactic dehydrogenase hinders the growth of human hepatocellular carcinoma cell lines.

BACKGROUND/AIMS: by reducing the number of ATP molecules produced via aerobic glycolysis, the inhibition of lactic dehydrogenase (LDH) should hinder the growth of neoplastic cells without damaging the normal cells which do not rely on this metabolic pathway for their energetic needs. Here, we studied the effect of oxamic and tartronic acids, 2 inhibitors of LDH, on aerobic glycolysis and cell replication of HepG2 and PLC/PRF/5 cells, 2 lines from human hepatocellular carcinomas. METHODS: aerobic glycolysis was measured by calculating the amounts of lactic acid formed. The effect on replication was assessed by culturing the cells in both standard conditions and glucose-deprived medium, which was used to shut down aerobic glycolysis. RESULTS: the oxamic and tartronic acids inhibited aerobic glycolysis, impaired the growth of both cell lines and also induced an increased expression of p53-upregulated modulator of apoptosis, a signal of cell death. A strong impairment of cell replication by oxamic acid was only found when the cells were cultured in the presence of glucose, indicating that it was for the most part owing to inhibition of aerobic glycolysis. CONCLUSIONS: inhibition of aerobic glycolysis achieved by blocking LDH could be useful in the treatment of human hepatocellular carcinomas. Without interfering with glucose metabolism in normal cells, it could hinder cell growth by itself and could also enhance the chemotherapeutic index of associated anticancer agents by decreasing the levels of ATP selectively in neoplastic cells.

Recognition of (2S)-aminomalonyl-acyl carrier protein (ACP) and (2R)-hydroxymalonyl-ACP by acyltransferases in zwittermicin A biosynthesis.

Polyketide synthases elongate a polyketide backbone by condensing carboxylic acid precursors that are thioesterified to either coenzyme A or an acyl carrier protein (ACP). Two of the three known ACP-linked extender units, (2S)-aminomalonyl-ACP and (2R)-hydroxymalonyl-ACP, are found in the biosynthesis of the agriculturally important antibiotic zwittermicin A. We previously reconstituted the formation of (2S)-aminomalonyl-ACP and (2R)-hydroxymalonyl-ACP from the primary metabolites l-serine and 1,3-bisphospho-d-glycerate. In this report, we characterize the two acyltransferases involved in the specific transfer of the (2S)-aminomalonyl and (2R)-hydroxymalonyl moieties from the ACPs associated with extender unit formation to the ACPs integrated into the polyketide synthase. This work establishes which acyltransferase recognizes each extender unit and also provides insight into the substrate selectivity of these enzymes. These are important step toward harnessing these rare polyketide synthase extender units for combinatorial biosynthesis.

Stopped-flow studies of the reaction of D-tartronate semialdehyde-2-phosphate with human neuronal enolase and yeast enolase 1.

We determined the kinetics of the reaction of human neuronal enolase and yeast enolase 1 with the slowly-reacting chromophoric substrate D-tartronate semialdehyde phosphate (TSP), each in tris (tris (hydroxymethyl) aminomethane) and another buffer at several Mg2+ concentrations, 50 or 100 microM, 1 mM and 30 mM. All data were biphasic, and could be satisfactorily fit, assuming either two successive first-order reactions or two independent first-order reactions. Higher Mg2+ concentrations reduce the relative magnitude of the slower reaction. The results are interpreted in terms of a catalytically significant interaction between the two subunits of these enzymes.

Enzymatic function of loop movement in enolase: preparation and some properties of H159N, H159A, H159F, and N207A enolases.

The hypothesis that His159 in yeast enolase moves on a polypeptide loop to protonate the phosphoryl of 2-phosphoglycerate to initiate its conversion to phosphoenolpyruvate was tested by preparing H159N, H159A, and H159F enolases. These have 0.07%-0.25% of the native activity under standard assay conditions and the pH dependence of maximum velocities of H159A and H159N mutants is markedly altered. Activation by Mg2+ is biphasic, with the smaller Mg2+ activation constant closer to that of the "catalytic" Mg2+ binding site of native enolase and the larger in the mM range in which native enolase is inhibited. A third Mg2+ may bind to the phosphoryl, functionally replacing proton donation by His159. N207A enolase lacks an intersubunit interaction that stabilizes the closed loop(s) conformation when 2-phosphoglycerate binds. It has 21% of the native activity, also exhibits biphasic Mg2+ activation, and its reaction with the aldehyde analogue of the substrate is more strongly inhibited than is its normal enzymatic reaction. Polypeptide loop(s) closure may keep a proton from His159 interacting with the substrate phosphoryl oxygen long enough to stabilize a carbanion intermediate.

Crystallographic studies on Ascaris suum NAD-malic enzyme bound to reduced cofactor and identification of an effector site.

The crystal structure of the mitochondrial NAD-malic enzyme from Ascaris suum, in a quaternary complex with NADH, tartronate, and magnesium has been determined to 2.0-A resolution. The structure closely resembles the previously determined structure of the same enzyme in binary complex with NAD. However, a significant difference is observed within the coenzyme-binding pocket of the active site with the nicotinamide ring of NADH molecule rotating by 198 degrees over the C-1-N-1 bond into the active site without causing significant movement of the other catalytic residues. The implications of this conformational change in the nicotinamide ring to the catalytic mechanism are discussed. The structure also reveals a binding pocket for the divalent metal ion in the active site and a binding site for tartronate located in a highly positively charged environment within the subunit interface that is distinct from the active site. The tartronate binding site, presumably an allosteric site for the activator fumarate, shows striking similarities and differences with the activator site of the human NAD-malic enzyme that has been reported recently. Thus, the structure provides additional insights into the catalytic as well as the allosteric mechanisms of the enzyme.

Molecular mechanism for the regulation of human mitochondrial NAD(P)+-dependent malic enzyme by ATP and fumarate.

The regulation of human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD-ME) by ATP and fumarate may be crucial for the metabolism of glutamine for energy production in rapidly proliferating tissues and tumors. Here we report the crystal structure at 2.2 A resolution of m-NAD-ME in complex with ATP, Mn2+, tartronate, and fumarate. Our structural, kinetic, and mutagenesis studies reveal unexpectedly that ATP is an active-site inhibitor of the enzyme, despite the presence of an exo binding site. The structure also reveals the allosteric binding site for fumarate in the dimer interface. Mutations in this binding site abolished the activating effects of fumarate. Comparison to the structure in the absence of fumarate indicates a possible molecular mechanism for the allosteric function of this compound.

Relationships between inhibition constants, inhibitor concentrations for 50% inhibition and types of inhibition: new ways of analysing data.

The concentration of an inhibitor that decreases the rate of an enzyme-catalysed reaction by 50%, symbolized i(0.5), is often used in pharmacological studies to characterize inhibitors. It can be estimated from the common inhibition plots used in biochemistry by means of the fact that the extrapolated inhibitor concentration at which the rate becomes infinite is equal to -i(0.5). This method is, in principle, more accurate than comparing the rates at various different inhibitor concentrations, and inferring the value of i(0.5) by interpolation. Its reciprocal, 1/i(0.5), is linearly dependent on v(0)/V, the uninhibited rate divided by the limiting rate, and the extrapolated value of v(0)/V at which 1/i(0.5) is zero allows the type of inhibition to be characterized: this value is 1 if the inhibition is strictly competitive; greater than 1 if the inhibition is mixed with a predominantly competitive component; infinite (i.e. 1/i(0.5) does not vary with v(0)/V) if the inhibition is pure non-competitive (i.e. mixed with competitive and uncompetitive components equal); negative if the inhibition is mixed with a predominantly uncompetitive component; and zero if it is strictly uncompetitive. The type of analysis proposed has been tested experimentally by examining inhibition of lactate dehydrogenase by oxalate (an uncompetitive inhibitor with respect to pyruvate) and oxamate (a competitive inhibitor with respect to pyruvate), and of cytosolic malate dehydrogenase by hydroxymalonate (a mixed inhibitor with respect to oxaloacetate). In all cases there is excellent agreement between theory and experiment.

Substrate and cofactor binding to fluorescently labeled cytoplasmic malate dehydrogenase.

Cytoplasmic malate dehydrogenase (cMDH) is a key enzyme in several metabolic pathways. Though its activity has been examined extensively, there are lingering mechanistic uncertainties involving substrate and cofactor binding. To more completely understand this enzyme's interactions with cofactor and substrate ligands, a fluorescent reporter group was introduced into the enzyme's structure. This was accomplished by selective modification of Cys 110. The reaction placed an aminonaphthaline sulfonic acid group near the enzyme's active site. Substrate, inhibitor, and NAD binding activities were characterized using changes in this label's fluorescence. Results demonstrated that both substrate and cofactor molecules bound to the enzyme in the absence of their companion ligands. This is in contrast to strictly ordered cofactor then substrate binding as has been suggested by kinetic analyses of closely related enzymes. Binding results also indicated that the cofactor, NAD, bound to cMDH in a negatively cooperative manner, but substrates and the inhibitor, hydroxymalonate, bound non-cooperatively. Multiple substrate binding modes were identified and interactions between substrate and cofactor binding were found.

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

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.