2-Deoxyribose 5-phosphate aldolase: Genetic analyses of structure

A total of 33 mutant strains of Salmonella typhimurium deficient in deoxyribose 5-phosphate activity have been isolated and characterized as missense or nonsense. Three-factor transductional analyses of the mutants were used to construct a fine structure map of the deoC gene, which codes for a peptide of 28,500 molecular weight. An unusual clustering of the missense mutants was observed, where 75% of all the missense mutants mapped in an area which corresponds to 19% of the total gene length. It is suggested that this area of the protein is particularly sensitive to amino acid replacements but that other areas of the protein are reasonably tolerant of such changes. Nonsense mutations are found scattered throughout the gene. This is expected since the carboxyl-terminal tyrosine is essential for enzymatic activity.     

Efficient synthesis of (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranose by using new 2-deoxy-d-ribose-5-phosphate aldolase from Streptococcus suis with moderate activity and aldehyde tolerance

The enzyme 2-deoxy-d-ribose-5-phosphate aldolase (DERA) is a useful tool for synthesizing statin side-chain intermediates. In this work, we identified the DERA from Streptococcus suis (SsDERA) by structural and sequence alignment and highly expressed it in Escherichia coli BL21. The recombinant SsDERA had a specific activity of 18.2 U mg⁻¹, K_M of 0.8 mM, and V_max of 32.9 μmol min⁻¹ mg⁻¹ toward 2-deoxy-d-ribose-5-phosphate under the optimal conditions: 40 °C and pH 7.0. The enzyme retained 23.3 % activity after incubation in 200 mM acetaldehyde for 2 h and 58.2 % activity in 100 mM chloroacetaldehyde for 2 h. The enzyme showed moderate activity and aldehyde tolerance compared with reported DERAs. The SsDERA-catalyzed reaction between 200 mM acetaldehyde and 100 mM chloroacetaldehyde generated (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranose in 76 % yield in 8 h. This work provides a new DERA for the synthesis of (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranose, which is a potential candidate for the industrial synthesis of statin intermediates.     

Kinetic mechanism of fuculose-1-phosphate aldolase from the hyperthermophilic archaeon Methanococcus jannaschii

Fuculose-1-phosphate aldolase (FucA) is a useful biocatalyst with potential applications in chiral synthesis. In this study, the overall kinetic mechanism of FucA from the archaeon Methanococcus jannaschii was studied. The K(m) values of dihydroxyacetone phosphate (DHAP) and dl-glyceraldehyde were 0.09 and 0.74 mM, respectively. Dead-end inhibition by trimethyl phosphonoacetate and dl-threose were competitive and uncompetitive with respect to DHAP and dl-glyceraldehyde. Inhibition patterns obtained using reaction products were noncompetitive vs. DHAP and competitive vs. dl-glyceraldehyde. The equilibrium constant was 8.309×10(-3) M as assessed by varying the [DHAP]/[product] ratio at a fixed dl-glyceraldehyde concentration and by measuring the change in DHAP concentration after equilibrium was reached. This constant is consistent with the K(eq) value obtained from (13)C NMR (15.625×10(-3) M). The resultant inhibition kinetics may suggest the insights of kinetic mechanism of the FucA catalyzed reaction.     

2-Deoxy-d-ribose-5-phosphate aldolase (DERA): applications and modifications

2-Deoxy-d-ribose-5-phosphate aldolase (DERA) is a class I aldolase that offers access to several building blocks for organic synthesis. It catalyzes the stereoselective C–C bond formation between acetaldehyde and numerous other aldehydes. However, the practical application of DERA as a biocatalyst is limited by its poor tolerance towards industrially relevant concentrations of aldehydes, in particular acetaldehyde. Therefore, the development of proper experimental conditions, including protein engineering and/or immobilization on appropriate supports, is required. The present review is aimed to provide a brief overview of DERA, its history, and progress made in understanding the functioning of the enzyme. Furthermore, the current understanding regarding aldehyde resistance of DERA and the various optimizations carried out to modify this property are discussed.

     

Directed evolution of an industrial biocatalyst: 2-deoxy-D-ribose 5-phosphate aldolase

Aldolases are emerging as powerful and cost efficient tools for the industrial synthesis of chiral molecules. They catalyze enantioselective carbon-carbon bond formations, generating up to two chiral centers under mild reaction conditions. Despite their versatility, narrow substrate ranges and enzyme inactivation under synthesis conditions represented major obstacles for large-scale applications of aldolases. In this study we applied directed evolution to optimize Escherichia coli 2-deoxy-D-ribose 5-phosphate aldolase (DERA) as biocatalyst for the industrial synthesis of (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranoside. This versatile chiral precursor for vastatin drugs like Lipitor (atorvastatin) is synthesized by DERA in a tandem-aldol reaction from chloroacetaldehyde and two acetaldehyde equivalents. However, E. coli DERA shows low affinity to chloroacetaldehyde and is rapidly inactivated at aldehyde concentrations useful for biocatalysis. Using high-throughput screenings for chloroacetaldehyde resistance and for higher productivity, several improved variants have been identified. By combination of the most beneficial mutations we obtained a tenfold improved variant compared to wild-type DERA with regard to (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranoside synthesis, under industrially relevant conditions.     

Characterization and application of a newly synthesized 2-deoxyribose-5-phosphate aldolase

A codon-optimized 2-deoxyribose-5-phosphate aldolase (DERA) gene was newly synthesized and expressed in Escherichia coli to investigate its biochemical properties and applications in synthesis of statin intermediates. The expressed DERA was purified and characterized using 2-deoxyribose-5-phosphate as the substrate. The specific activity of recombinant DERA was 1.8 U/mg. The optimum pH and temperature for DERA activity were pH 7.0 and 35 °C, respectively. The recombinant DERA was stable at pH 4.0–7.0 and at temperatures below 50 °C. The enzyme activity was inhibited by 1 mM of Ni²⁺, Ba²⁺ and Fe²⁺. The apparent K _m and V _max values of purified enzyme for 2-deoxyribose-5-phosphate were 0.038 mM and 2.9 μmol min^?1 mg^?1, for 2-deoxyribose were 0.033 mM and 2.59 μmol min^?1 mg^?1, respectively, which revealed that the enzyme had similar catalytic efficiency towards phosphorylated and non-phosphorylated substrates. To synthesize statin intermediates, the bioconversion process for production of (3R, 5S)-6-chloro-2,4,6-trideoxyhexose from chloroacetaldehyde and acetaldehyde by the recombinant DERA was developed and a conversion of 94.4 % was achieved. This recombinant DERA could be a potential candidate for application in production of (3R, 5S)-6-chloro-2,4,6-trideoxyhexose.     

Cloning and characterisation of a new 2-deoxy-D-ribose-5-phosphate aldolase from Rhodococcus erythropolis

A new 2-deoxy-D-ribose-5-phoshate aldolase (DERA) gene was cloned from Rhodococcus erythropolis strain DSM 311, recombinantly expressed in Escherichia coli, and purified via affinity chromatography which yielded a homo-dimeric enzyme of 44.3 kDa as apparent by size exclusion chromatography. To characterise the enzyme, investigations about pH and temperature tolerance, stability, as well as analyses on resistance to organic solvents and acetaldehyde were performed. In addition, kinetic constants of the new DERA(RE) were compared to respective values of the DERA from E. coli (DERA(EC)). Stability of DERA(RE) turned out to be a crucial factor: The pH for optimal DERA(RE) activity was determined to be 7.0, whereas the highest stability was achieved at pH 9.0 with a half-life of approximately 20 days. The optimal temperature for DERA(RE) activity was 65 °C, but coupled with a rather low stability (half-life of 2 min). The highest stability was achieved at 25 °C. The new enzyme exhibits high resistance to organic solvents and acetaldehyde with a half-life being 2.5× higher compared to DERA(EC) under the exposure of 300 mM acetaldehyde. Hence it has the potential as a new promising biocatalyst with applications in organic synthesis.     

Structure-based mutagenesis approaches toward expanding the substrate specificity of D-2-deoxyribose-5-phosphate aldolase

2-Deoxyribose-5-phosphate aldolase (DERA, EC 4.1.2.4) catalyzes the reversible aldol reaction between acetaldehyde and D-glyceraldehyde-3-phosphate to generate D-2-deoxyribose-5-phosphate. It is unique among the aldolases as it catalyzes the reversible asymmetric aldol addition reaction of two aldehydes. In order to expand the substrate scope and stereoselectivity of DERA, structure-based substrate design as well as site-specific mutation has been investigated. Using the 1.05 A crystal structure of DERA in complex with its natural substrate as a guide, five site-directed mutants were designed in order to improve its activity with the unnatural nonphosphorylated substrate, D-2-deoxyribose. Of these, the S238D variant exhibited a 2.5-fold improvement over the wild-type enzyme in the retroaldol reaction of 2-deoxyribose. Interestingly, this S238D mutant enzyme was shown to accept 3-azidopropinaldehyde as a substrate in a sequential asymmetric aldol reaction to form a deoxy-azidoethyl pyranose, which is a precursor to the corresponding lactone and the cholesterol-lowering agent Lipitor. This azidoaldehyde is not a substrate for the wild-type enzyme. Another structure-based design of new nonphosphorylated substrates was focused on the aldol reaction with inversion in enantioselectivity using the wild type or the S238D variant as the catalyst and 2-methyl-substituted aldehydes as substrates. An example was demonstrated in the asymmetric synthesis of a deoxypyranose as a new effective synthon for the total synthesis of epothilones. In addition, to facilitate the discovery of new enzymatic reactions, the engineered E. coli strain SELECT (Deltaace, adhC, DE3) was developed to be used in the future for selection of DERA variants with novel nonphosphorylated acceptor specificity.     

Presence of a novel phosphopentomutase and a 2-deoxyribose 5-phosphate aldolase reveals a metabolic link between pentoses and central carbon metabolism in the hyperthermophilic archaeon Thermococcus kodakaraensis

Numerous bacteria and mammalian cells harbor two enzymes, phosphopentomutase (PPM) and 2-deoxyribose 5-phosphate aldolase (DERA), involved in the interconversion between nucleosides and central carbon metabolism. In this study, we have examined the presence of this metabolic link in the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1. A search of the genome sequence of this strain revealed the presence of a closely related orthologue (TK2104) of bacterial DERA genes while no orthologue related to previously characterized PPM genes could be detected. Expression, purification, and characterization of the TK2104 protein product revealed that this gene actually encoded a DERA, catalyzing the reaction through a class I aldolase mechanism. As PPM activity was detected in T. kodakaraensis cells, we partially purified the protein to examine its N-terminal amino acid sequence. The sequence corresponded to a gene (TK1777) similar to phosphomannomutases within COG1109 but not COG1015, which includes all previously identified PPMs. Heterologous gene expression of TK1777 and characterization of the purified recombinant protein clearly revealed that the gene indeed encoded a PPM. Both enzyme activities could be observed in T. kodakaraensis cells under glycolytic and gluconeogenic growth conditions, whereas the addition of ribose, 2-deoxyribose, and 2'-deoxynucleosides in the medium did not lead to a significant induction of these activities. Our results clearly indicate the presence of a metabolic link between pentoses and central carbon metabolism in T. kodakaraensis, providing an alternative route for pentose biosynthesis through the functions of DERA and a structurally novel PPM.     

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.     

The sterochemistry at carbon 3 of pyruvate lyase condensation products. 2-Keto-3-deoxygluconate 6-phosphate and 2-keto-3-deoxygalactonate-6-phosphate aldolase of Pseudomonas saccharophila.

In Pseudomonas saccharophila 2-keto-3-deoxygalactonate-6-P aldolase (EC 4.1.2.21) is induced by growth on galatose while 2-keto-3-deoxygluconate-6-P aldolase (EC 4.1.2.14) is constitutive. These enzymes catalyze identical reactions except for the configuration fixed at C-4 during the condensation reaction. It was found with each enzyme that in a condensation between [3-3H3]pyruvate and D-glyceraldehyde-3-P, the respective condensation products were formed 8 to 10 times faster than tritium was released to water. Since pyruvate deprotonation is obligatory for condensation, the above result requires a hydrogen isotope effect in enolpyruvate formation, which must be then at least partially rate limiting for C--C synthesis. Further, condensation between D-glyceraldehyde-3-P and (3R)-[3-3H, 2H,H]pyruvate or (3S)-[3-3H, 2H,H]pyruvate, as catalyzed by each enzyme, enriched for (3R)- and (3S)-3-3H, 2H-labeled condensation product, respectively. Thus, each enzyme catalyzes C--C and C--H synthesis with retention of configuration at C-3. This shows that the active sites of both enzymes are asymmetric since solutes can only approach a single face of the bound pyruvyl enolate. In addition, the respective aldehyde specific portions of the two active sites must have opposite chiralities, with respect to each other, for correctly orienting the carbonyl faces of the incoming D-glyceraldehyde-3-P, to generate the correct configuration at C-4 of the respective condensation products.     

Mechanism for folate-independent aldolase reaction catalyzed by serine hydroxymethyltransferase

Serine hydroxymethyltransferase catalyzes the cleavage of β-hydroxyamino acids into glycine and aldehydes in the absence of tetrahydrofolate. The enzyme accepts various β-hydroxyamino acids as the substrate of this reaction. The reaction rate varies depending on the substituent and stereochemistry at the Cβ atom: the erythro forms and the β-phenyl substituent are preferred over the threo forms and the β-methyl substituent, respectively. Although several mechanisms have been proposed, what determines the substrate preference remains unclear. We first performed quantum mechanical calculations to assess the validity of the reaction mechanisms. The results indicate that the retro-aldol mechanism starting with abstraction of the proton from the β-hydroxyl group is plausible. This also suggests that Cα-Cβ bond cleavage is the rate-limiting step. We next measured the dependence of the rate constants on temperature with four representative substrates and calculated the activation energies and pre-exponential factors from the Arrhenius plots. The activation energies of the erythro forms were lower than those of the threo forms. The β-phenyl substituent lowered the activation energy in the threo form, whereas it did not alter the activation energy but increased the pre-exponential factor in the erythro form. We present a unified model to explain the origin of the substituent and stereochemical preferences by combining the theoretical and experimental results. A possible biological role of the tetrahydrofolate-independent activity in thermophiles is also discussed.     

Deoxyribose 5-phosphate aldolase of Bacillus cereus: purification and properties.

Deoxyribose 5-phosphate aldolase was purified 41 times from Bacillus cereus induced by growth on deoxyribonucleosides. The purification procedure includes ammonium sulphate fractionation, gel filtration on Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel and preparative electrophoresis on 10% polyacrylamide gel. The enzyme is stable above pH 6.5, but is rapidly inactivated by sulfhydryl reagents. Being insensitive to EDTA, it may be considered as a Class I aldolase. Among a number of compounds tested (including some carboxylic acids, free and phosphorylated pentoses, nucleotides and nucleosides), none has been found to affect the enzyme activity. The enzyme appears to be dimeric, with a subunit Mr of 23,600. A Km of 4.4 x 10(-4) M was calculated for dRib 5-P.     

来自Staphylococcus aureus N315的2-脱氧-D-核糖5-磷酸醛缩酶的工程菌构建、表达纯化与性质鉴定

利用基因挖掘在数据库中发现一种来自Staphylococcus aureus N315的潜在DERA(SaDERA),将其基因密码子优化后实现了在大肠杆菌中的表达,重组酶经纯化后研究了其催化性质。结果表明:构建的工程菌具有较高的可溶表达量(占总蛋白的70%),通过简单的一步纯化即可得到电泳纯的酶;SaDERA是一种同源二聚体的酶(5.7×104),其最适反应条件是pH 7.7和45℃;SaDERA具有良好的碱耐受性,在pH 11.0、25℃的条件下温浴24 h后仍有93%的残余活力;SaDERA具有良好的乙醛耐受性,在0.3 mol/L乙醛浓度、25℃下,30 min内保持了70%以上的残余活力;乙醛连续自缩合产物被纯化并鉴定,所得产品为两次缩合产物。     

Analysis of the class I aldolase binding site architecture based on the crystal structure of 2-deoxyribose-5-phosphate aldolase at 0.99A resolution

The crystal structure of the bacterial (Escherichia coli) class I 2-deoxyribose-5-phosphate aldolase (DERA) has been determined by Se-Met multiple anomalous dispersion (MAD) methods at 0.99A resolution. This structure represents the highest-resolution X-ray structure of an aldolase determined to date and enables a true atomic view of the enzyme. The crystal structure shows the ubiquitous TIM alpha/beta barrel fold. The enzyme contains two lysine residues in the active site. Lys167 forms the Schiff base intermediate, whereas Lys201, which is in close vicinity to the reactive lysine residue, is responsible for the perturbed pK(a) of Lys167 and, hence, also a key residue in the reaction mechanism. DERA is the only known aldolase that is able to use aldehydes as both aldol donor and acceptor molecules in the aldol reaction and is, therefore, of particular interest as a biocatalyst in synthetic organic chemistry. The uncomplexed DERA structure enables a detailed comparison with the substrate complexes and highlights a conformational change in the phosphate-binding site. Knowledge of the enzyme active-site environment has been the basis for exploration of catalysis of non-natural substrates and of mutagenesis of the phosphate-binding site to expand substrate specificity. Detailed comparison with other class I aldolase enzymes and DERA enzymes from different organisms reveals a similar geometric arrangement of key residues and implies a potential role for water as a general base in the catalytic mechanism.     

Catalysis and binding in L-ribulose-5-phosphate 4-epimerase: a comparison with L-fuculose-1-phosphate aldolase.

L-Ribulose-5-phosphate (L-Ru5P) 4-epimerase and L-fuculose-1-phosphate (L-Fuc1P) aldolase are evolutionarily related enzymes that display 26% sequence identity and a very high degree of structural similarity. They both employ a divalent cation in the formation and stabilization of an enolate during catalysis, and both are able to deprotonate the C-4 hydroxyl group of a phosphoketose substrate. Despite these many similarities, subtle distinctions must be present which allow the enzymes to catalyze two seemingly different reactions and to accommodate substrates differing greatly in the position of the phosphate (C-5 vs C-1). Asp76 of the epimerase corresponds to the key catalytic acid/base residue Glu73 of the aldolase. The D76N mutant of the epimerase retained considerable activity, indicating it is not a key catalytic residue in this enzyme. In addition, the D76E mutant did not show enhanced levels of background aldolase activity. Mutations of residues in the putative phosphate-binding pocket of the epimerase (N28A and K42M) showed dramatically higher values of K(M) for L-Ru5P. This indicates that both enzymes utilize the same phosphate recognition pocket, and since the phosphates are positioned at opposite ends of the respective substrates, the two enzymes must bind their substrates in a reversed or "flipped" orientation. The epimerase mutant D120N displays a 3000-fold decrease in the value of k(cat), suggesting that Asp120' provides a key catalytic acid/base residue in this enzyme. Analysis of the D120N mutant by X-ray crystallography shows that its structure is indistinguishable from that of the wild-type enzyme and that the decrease in activity was not simply due to a structural perturbation of the active site. Previous work [Lee, L. V., Poyner, R. R., Vu, M. V., and Cleland, W. W. (2000) Biochemistry 39, 4821-4830] has indicated that Tyr229' likely provides the other catalytic acid/base residue. Both of these residues are supplied by an adjacent subunit. Modeling of L-Ru5P into the active site of the epimerase structure suggests that Tyr229' is responsible for deprotonating L-Ru5P and Asp120' is responsible for deprotonating its epimer, D-Xu5P.