Identification of lactaldehyde dehydrogenase in Methanocaldococcus jannaschii and its involvement in production of lactate for F420 biosynthesis

One of the early steps in the biosynthesis of coenzyme F(420) in Methanocaldococcus jannaschii requires generation of 2-phospho-L-lactate, which is formed by the phosphorylation of L-lactate. Preliminary studies had shown that L-lactate in M. jannaschii is not derived from pyruvate, and thus an alternate pathway(s) for its formation was examined. Here we report that L-lactate is formed by the NAD(+)-dependent oxidation of l-lactaldehyde by the MJ1411 gene product. The lactaldehyde, in turn, was found to be generated either by the NAD(P)H reduction of methylglyoxal or by the aldol cleavage of fuculose-1-phosphate by fuculose-1-phosphate aldolase, the MJ1418 gene product.     

Performance of an immobilized fuculose-1-phosphate aldolase for stereoselective synthesis

A derivative of fuculose-1-phosphate aldolase, immobilized with high loading on glyoxal–agarose gels, has been characterized and evaluated as a biocatalyst for an aldol addition reaction. The reaction of the solid biocatalyst was diffusion-controlled for conversion of its natural substrate. Nevertheless, when catalyzing the synthesis of a biologically active aminopolyol, the lower reaction rate with non-natural substrates led to a process controlled by the intrinsic enzyme kinetics. The resulting biocatalyst has high synthetic specific activity and has been successfully used in batch synthesis reactions with high conversion. In addition, the immobilized aldolase has been employed in fed-batch synthesis, increasing the selectivity of the reaction and obtaining high conversion (88%).     

Influence of secondary reactions on the synthetic efficiency of DHAP-aldolases

The effect of secondary reactions on DHAP-dependent aldolase stereoselective synthesis yields is reported. The fuculose-1-phosphate aldolase catalyzed synthesis between DHAP and Cbz-S-alaninal has been chosen as case study. It has been demonstrated that DHAP is not only chemically degraded in the reaction medium, but also enzymatically. The last reaction has been shown to take place when type II aldolases are used as biocatalysts. In order to minimize the effect of non-desired reactions, temperature reduction has been shown to be favorable, and operation at 4 degrees C has been chosen as appropriate. On the other hand, the fed-batch addition of DHAP also increased the synthesis yields and, combined with low temperature, led to almost quantitative conversion.     

Characterization of alditol oxidase from Streptomyces coelicolor and its application in the production of rare sugars

A synthetic platform for the cascade synthesis of rare sugars using Escherichia coli whole cells was established. In the cascade, the donor substrate dihydroxyacetone phosphate (DHAP) was generated from glycerol by glycerol kinase (GK) and glycerol phosphate oxidase (GPO). The acceptor d-glyceraldehyde was directly produced from glycerol by an alditol oxidase. Then, the aldol reaction between DHAP and d-glyceraldehyde was performed by l-rhamnulose-1-phosphate aldolase (RhaD) to generate the corresponding sugar-1-phosphate. Finally, the phosphate group was removed by fructose-1-phosphatase (YqaB) to obtain the rare sugars d-sorbose and d-psicose. To accomplish this goal, the alditol oxidase from Streptomyces coelicolor (AldO_S.coe) was expressed in E. coli and the purified AldO_S.coe was characterized. Furthermore, a recombinant E. coli strain overexpressing six enzymes including AldO_S.coe was constructed. Under the optimized conditions, it produced 7.9 g/L of d-sorbose and d-psicose with a total conversion rate of 17.7% from glycerol. This study provides a useful and cost-effective method for the synthesis of rare sugars.     

Structural and Functional Analysis of Fucose-Processing Enzymes from Streptococcus pneumoniae

Fucose metabolism pathways are present in many bacterial species and typically contain the central fucose-processing enzymes fucose isomerase (FcsI), fuculose kinase (FcsK), and fuculose-1-phosphate aldolase (FcsA). Fucose initially undergoes isomerization by FcsI producing fuculose, which is then phosphorylated by FcsK. FcsA cleaves the fuculose-1-phosphate product into lactaldehyde and dihydroxyacetone phosphate, which can be incorporated into central metabolism allowing the bacterium to use fucose as an energy source. Streptococcus pneumoniae has fucose-processing operons containing homologs of FcsI, FcsK, and FcsA; however, this bacterium appears unable to utilize fucose as an energy source. To investigate this contradiction, we performed biochemical and structural studies of the S. pneumoniae fucose-processing enzymes SpFcsI, SpFcsK, and SpFcsA. These enzymes are demonstrated to act in a sequential manner to ultimately produce dihydroxyacetone phosphate and have structural features entirely consistent with their observed biochemical activities. Analogous to the regulation of the Escherichia coli fucose utilization operon, fuculose-1-phosphate appears to act as an inducing molecule for activation of the S. pneumoniae fucose operon. Despite our evidence that S. pneumoniae appears to have the appropriate regulatory and biochemical machinery for fucose metabolism, we confirmed the inability of the S. pneumoniae TIGR4 strain to grow on fucose or on the H-disaccharide, which is the probable substrate of the transporter for the pathway. On the basis of these observations, we postulate that the S. pneumoniae fucose-processing pathway has a non-metabolic role in the interaction of this bacterium with its human host.     

Kinetic modelling of aldolase-catalyzed addition between dihydroxyacetone phosphate and (S)-alaninal

This paper is focused on the development of a kinetic model for an aldolase-catalyzed reaction. The aldol addition between dihydroxyacetone phosphate (DHAP) and (S)-benzyloxycarbonyl-alaninal ((S)-Cbz-alaninal) catalyzed by the four DHAP-dependent aldolases is a promising way for the synthesis of four complementary diastereoisomers with potential biological activity. The reaction catalyzed by fuculose-1-phosphate aldolase (FucA) conducts to a synthesis product with a 100% diastereomeric excess. A kinetic model has been proposed including both the synthesis and a parallel non-desired secondary reaction. The model involved an ordered two-substrate mechanism for the synthesis and non-competitive inhibition by (S)-Cbz-alaninal and competitive inhibition by methylglyoxal byproduct in both reactions. The values of the model kinetic parameters were determined and the model validated in batch and fed-batch synthesis reactions. The obtained model could be extended to explain the behavior of other class II DHAP-dependent aldolases and exploited in simulation for reactor design purposes.     

A simple feedback control of Escherichia coli growth for recombinant aldolase production in fed-batch mode

Fed-batch production of recombinant fuculose-1-phosphate aldolase (FucA) by Escherichia coli XL1 Blue MRF′ (pTrcfuc) has been automated by using a simple feedback specific growth rate control strategy. Non-induced continuous cultures were conducted in order to characterize substrate consumption and carbon dioxide production yields and rates. In fed-batch cultures, substrate feeding rate was adjusted using on-line biomass estimation based on exhaust gas analysis and macroscopic mass balances. Overexpression of recombinant protein induced by isopropyl-β-d-thiogalactopyranoside (IPTG) under trc promoter did not affect significantly the control of specific growth rate during 7 h after induction. Growth and protein production curves were parallel until high level of protein expression started to inhibit cell growth. The proposed specific growth rate control strategy has been successfully applied to both non-induced and induced fed-batch cultures that do not exhibit severe growth rate depression.     

Stereochemical specificity of the biosynthesis of the alkyl ether bond in alkyl ether lipids.

The stereochemical course of the formation of the alkyl ether bond in alkyl ether lipids was investigated through the synthesis of stereospecifically labeled acyl R- or S-[1-3H]dihydroxyacetone 3-phosphate (DHAP) starting from L-glyceraldehyde. It was demonstrated directly that the formation of the alkyl ether bond results in the stereospecific exchange of the pro-R C-1 hydrogen of DHAP with a proton of water. The configuration of the hydrogen that is retained on C-1 after formation of the alkyl ether bond was also investigated. The alkyl ether lipid was degraded, and the DHAP backbone isolated as glycerol, converted to DHAP via glycerol 3-phosphate and treated with either aldolase or triose phosphate isomerase. The results demonstrated that the retained hydrogen on C-1, which was pro-S in the starting substrate, was pro-S in the product alkyl ether.     

Dihydroxyacetone synthase from a methanol-utilizing carboxydobacterium, Acinetobacter sp. strain JC1 DSM 3803.

Acinetobacter sp. strain JC1 DSM 3803, a carboxydobacterium, grown on methanol was found to show dihydroxyacetone synthase, dihydroxyacetone kinase, and ribulose 1,5-bisphosphate carboxylase, but no hydroxypyruvate reductase and very low hexulose 6-phosphate synthase, activities. The dihydroxyacetone synthase was found to be expressed earlier than the ribulose 1,5-bisphosphate carboxylase. The dihydroxyacetone synthase was purified 19-fold in eight steps to homogeneity, with a yield of 9%. The final specific activity of the purified enzyme was 1.12 micromol of NADH oxidized per min per mg of protein. The molecular weight of the native enzyme was determined to be 140,000. Sodium dodecyl sulfate-gel electrophoresis revealed a subunit of molecular weight 73,000. The optimum temperature and pH were 30 degrees C and 7.0, respectively. The enzyme was inactivated very rapidly at 70 degrees C. The enzyme required Mg2+ and thiamine pyrophosphate for maximal activity. Xylulose 5-phosphate was found to be the best substrate when formaldehyde was used as a glycoaldehyde acceptor. Erythrose 4-phosphate, glycolaldehyde, and formaldehyde were found to act as excellent substrates when xylulose 5-phosphate was used as a glycoaldehyde donor. The Kms for formaldehyde and xylulose 5-phosphate were 1.86 mM and 33.3 microM, respectively. The enzyme produced dihydroxyacetone from formaldehyde and xylulose 5-phosphate. The enzyme was found to be expressed only in cells grown on methanol and shared no immunological properties with the yeast dihydroxyacetone synthase.     

Stereochemistry of nonnatural aldol reactions catalyzed by DHAP aldolases

A coupled enzymatic assay was developed for quantitative determination of the stereoisomeric products formed in aldol reactions catalyzed by dihydroxyacetone phosphate (DHAP)-dependent aldolases. Three of the four stereoisomers could be determined directly; the fourth one was calculated. This procedure is based on the reversibility of the aldol reaction and requires no derivatization or work-up of the product samples, only removal or inactivation of the biocatalyst. In comparison with other methods the enzymatic assay is highly accurate and fast. Determination of isomer formation with 10 different acceptor substrates applying this procedure gave unprecedented insight in the stereochemistry of fructose-1,6-bisphosphate aldolase from Staphylococcus carnosus and l-rhamnulose-1-phosphate aldolase from E. coli.     

Kinetic evidence for interaction between aldolase and D-glyceraldehyde-3-phosphate dehydrogenase.

The possibility of interaction between purified rabbit muscle aldolase and D-glyceraldehyde-3-phosphate dehydrogenase was studied by rapid kinetic methods, by analyzing the kinetics of the consecutive reaction catalyzed by the coupled enzyme system. The Km of the intermediary product, glyceraldehyde 3-phosphate, produced by aldolase was determined in the coupled reaction for glyceraldehyde-3-phosphate dehydrogenase. Its value corresponds to that of the aldehyde (active) form of glyceraldehyde 3-phosphate, although in the given conditions the aldehyde leads to diol interconversion is faster than the enzymic reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase. We suggest that above a certain concentration of the enzymes the glyceraldehyde 3-phosphate produced by aldolase gets direct access to glyceraldehyde-3-phosphate dehydrogenase without participating in the aldehyde leads to diol interconversion which otherwise would occur if the substrate were to mix with the bulk medium.     

Replacement of a phenylalanine by a tyrosine in the active site confers fructose-6-phosphate aldolase activity to the transaldolase of Escherichia coli and human origin

Based on a structure-assisted sequence alignment we designed 11 focused libraries at residues in the active site of transaldolase B from Escherichia coli and screened them for their ability to synthesize fructose 6-phosphate from dihydroxyacetone and glyceraldehyde 3-phosphate using a newly developed color assay. We found one positive variant exhibiting a replacement of Phe(178) to Tyr. This mutant variant is able not only to transfer a dihydroxyacetone moiety from a ketose donor, fructose 6-phosphate, onto an aldehyde acceptor, erythrose 4-phosphate (14 units/mg), but to use it as a substrate directly in an aldolase reaction (7 units/mg). With a single amino acid replacement the fructose-6-phosphate aldolase activity was increased considerably (>70-fold compared with wild-type). Structural studies of the wild-type and mutant protein suggest that this is due to a different H-bond pattern in the active site leading to a destabilization of the Schiff base intermediate. Furthermore, we show that a homologous replacement has a similar effect in the human transaldolase Taldo1 (aldolase activity, 14 units/mg). We also demonstrate that both enzymes TalB and Taldo1 are recognized by the same polyclonal antibody.     

The carbon assimilation pathways of Methylococcus capsulatus, Pseudomonas methanica and Methylosinus trichosporium (OB3B) during growth on methane

d-arabino-3-Hexulose 6-phosphate was prepared by condensation of formaldehyde with ribulose 5-phosphate in the presence of 3-hexulose phosphate synthase from methane-grown Methylococcus capsulatus. The 3-hexulose phosphate was unstable in solutions of pH greater than 3, giving a mixture of products in which, after dephosphorylation, allulose and fructose were detected. A complete conversion of d-ribulose 5-phosphate and formaldehyde into d-fructose 6-phosphate was demonstrated in the presence of 3-hexulose phosphate synthase and phospho-3-hexuloisomerase (prepared from methane-grown M. capsulatus). d-Allulose 6-phosphate was prepared from d-allose by way of d-allose 6-phosphate. No evidence was found for its metabolism by extracts of M. capsulatus, thus eliminating it as an intermediate in the carbon assimilation process of this organism. A survey was made of the enzymes involved in the regeneration of pentose phosphate during C(1) assimilation via a modified pentose phosphate cycle. On the basis of the presence of the necessary enzymes, two alternative routes for cleavage of fructose 6-phosphate are suggested, one route involves fructose diphosphate aldolase and the other 6-phospho-2-keto-3-deoxygluconate aldolase. A detailed formulation of the complete ribulose monophosphate cycle of formaldehyde fixation is presented. The energy requirements for carbon assimilation by this cycle are compared with those for the serine pathway and the ribulose diphosphate cycle of carbon dioxide fixation. A cyclic scheme for oxidation of formaldehyde via 6-phosphogluconate is suggested.     

Brownian dynamics simulations of aldolase binding glyceraldehyde 3-phosphate dehydrogenase and the possibility of substrate channeling

Brownian dynamics (BD) simulations test for channeling of the substrate, glyceraldehyde 3-phosphate (GAP), as it passes between the enzymes fructose-1,6-bisphosphate aldolase (aldolase) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). First, BD simulations determined the favorable complexes between aldolase and GAPDH; two adjacent subunits of GAPDH form salt bridges with two subunits of aldolase. These intermolecular contacts provide a strong electrostatic interaction between the enzymes. Second, BD simulates GAP moving out of the active site of the A or D aldolase subunit and entering any of the four active sites of GAPDH. The efficiency of transfer is determined as the relative number of BD trajectories that reached any active site of GAPDH. The distribution functions of the transfer time were calculated based on the duration of successful trajectories. BD simulations of the GAP binding from solution to aldolase/GAPDH complex were compared to the channeling simulations. The efficiency of transfer of GAP within an aldolase/GAPDH complex was 2 to 3% compared to 1.3% when GAP was binding to GAPDH from solution. There is a preference for GAP channeling between aldolase and GAPDH when compared to binding from solution. However, this preference is not large enough to be considered as a theoretical proof of channeling between these proteins.     

Biochemical and structural exploration of the catalytic capacity of Sulfolobus KDG aldolases

Aldolases are enzymes with potential applications in biosynthesis, depending on their activity, specificity and stability. In the present study, the genomes of Sulfolobus species were screened for aldolases. Two new KDGA [2-keto-3-deoxygluconate (2-oxo-3-deoxygluconate) aldolases] from Sulfolobus acidocaldarius and Sulfolobus tokodaii were identified, overexpressed in Escherichia coli and characterized. Both enzymes were found to have biochemical properties similar to the previously characterized S. solfataricus KDGA, including the condensation of pyruvate and either D,L-glyceraldehyde or D,L-glyceraldehyde 3-phosphate. The crystal structure of S. acidocaldarius KDGA revealed the presence of a novel phosphate-binding motif that allows the formation of multiple hydrogen-bonding interactions with the acceptor substrate, and enables high activity with glyceraldehyde 3-phosphate. Activity analyses with unnatural substrates revealed that these three KDGAs readily accept aldehydes with two to four carbon atoms, and that even aldoses with five carbon atoms are accepted to some extent. Water-mediated interactions permit binding of substrates in multiple conformations in the spacious hydrophilic binding site, and correlate with the observed broad substrate specificity.     

Dynamic interactions of enzymes involved in triosephosphate metabolism

A steady-state kinetic analysis of the coupled reactions catalysed by the three-enzyme system, aldolase, glyceraldehyde-3-phosphate dehydrogenase and triosephosphate isomerase, was performed. The kinetic parameters of the progress curves of end-product formation calculated for noninteracting enzymes were compared with those measured in the two-enzyme and three-enzyme systems. Changes in the fluorescence anisotropy of labelled dehydrogenase upon addition of aldolase and/or isomerase were also measured. Glyceraldehyde-3-phosphate oxidation catalysed by glyceraldehyde-3-phosphate dehydrogenase in the presence of isomerase (which ensures rapid equilibration of the triosephosphates) follows single first-order kinetics. The rate constant depends simply on the concentration of the dehydrogenase, indicating no kinetically significant isomerase-dehydrogenase interaction. Fluorescence anisotropy measurements also fail to reveal complex formation between the two enzymes. The steady-state velocity of 3-phosphoglycerate formation from fructose 1, 6-bisphosphate in the reactions catalysed by aldolase and dehydrogenase is not increased twofold on addition of the isomerase, even though a 1:2 stoichiometry of fructose 1,6-bisphosphate/glyceraldehyde 3-phosphate is expected. In fact, by increasing the concentration of the isomerase, the steady-state velocity actually decreases. This effect of the isomerase may be a kinetic consequence of an aldolase-isomerase interaction, which results in a decrease of aldolase activity. Furthermore, the fluorescence anisotropy of labelled dehydrogenase, measured at different aldolase concentrations, is significantly lower when the sample contains isomerase. The decrease in the steady-state velocity of the consecutive reactions caused by the elevation of isomerase concentration could be negated by increasing the dehydrogenase concentrations in the three-enzyme system. All of these observations fit the assumption that the amount of aldolase-dehydrogenase complex is reduced due to competition of isomerase with dehydrogenase. The alternate binding of dehydrogenase and isomerase to aldolase may regulate the flux rate of glycolysis.     

Mechanistic insight into 3-deoxy-D-manno-octulosonate-8-phosphate synthase and 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase utilizing phosphorylated monosaccharide analogues

Escherichia coli 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8-P) synthase is able to utilize the five-carbon phosphorylated monosaccharide, 2-deoxyribose 5-phosphate (2dR5P), as an alternate substrate, but not D-ribose 5-phosphate (R5P) nor the four carbon analogue D-erythrose 4-phosphate (E4P). However, E. coli KDO8-P synthase in the presence of either R5P or E4P catalyzes the rapid consumption of approximately 1 mol of PEP per active site, after which consumption of PEP slows to a negligible but measurable rate. The mechanism of this abortive utilization of PEP was investigated using [2,3-(13)C(2)]-PEP and [3-F]-PEP, and the reaction products were determined by (13)C, (31)P, and (19)F NMR to be pyruvate, phosphate, and 2-phosphoglyceric acid (2-PGA). The formation of pyruvate and 2-PGA suggests that the reaction catalyzed by KDO8-P synthase may be initiated via a nucleophilic attack to PEP by a water molecule. In experiments in which the homologous enzyme, 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7-P) synthase was incubated with D,L-glyceraldehyde 3-phosphate (G3P) and [2,3-(13)C(2)]-PEP, pyruvate and phosphate were the predominant species formed, suggesting that the reaction catalyzed by DAH7-P synthase starts with a nucleophilic attack by water onto PEP as observed in E. coli KDO8-P synthase.