Deoxyribose 5-phosphate Aldolase Research Articles

Characterization of a widespread sugar phosphate-processing bacterial microcompartment

Many prokaryotes form Bacterial Microcompartments (BMCs) that encapsulate segments of specialized metabolic pathways to enhance catalysis. The various functions of metabolosomes, catabolic BMCs, are dictated by the signature enzyme that processes initial substrates of the confined pathway. The components and native functions of several metabolosomes have been experimentally characterized; however one of the most prevalent across all bacteria has yet to be studied. Sugar Phosphate Utilizing (SPU) BMC loci encode enzymes predicted to be involved in sugar phosphate metabolism. The SPU genetic loci are found in organisms occupying habitats ranging from soils to hot springs, highlighting the ubiquity of the SPU BMC. We bioinformatically characterized seven SPU subtypes, all which contain an enzyme unique to SPU BMCs, a deoxyribose 5-phosphate aldolase (DERA). Here, we define the fundamental characteristics of SPU BMCs and have expressed, purified, and characterized a set of SPU core enzymes. These include a protein-protein complex formed between a SPU BMC DERA and a predicted ribose 5-phosphate isomerase. Further, we show that the SPU BMC DERA is catalytically active and propose that it acts as the universal signature enzyme for the SPU BMC, with implications for fundamental understanding and biotechnological applications of SPU BMCs.

An Aldolase-Based New Pathway for Bioconversion of Formaldehyde and Ethanol into 1,3-Propanediol in Escherichia coli

Formaldehyde (HCHO) is a reactive one-carbon compound that is interesting for biosynthesis. The assimilation of HCHO depends on the catalysis of aldolase. Here, we present a novel synthetic pathway in E.coli to convert HCHO and ethanol into 1,3-propanediol (PDO) using a deoxyribose-5-phosphate aldolase (DERA). DERA condenses HCHO and acetaldehyde to form 3-hydroxypropionaldehyde, the direct precursor of PDO formation. This new pathway opens up the possibility to synthesize an appealing C3 compound from a C1 compound and a C2 compound without carbon loss in contrast to all the other known PDO synthetic pathways where typically 30-50% of the carbons are lost as CO2 and other byproducts. The pathway is successfully demonstrated by elaborating three metabolic modules. First, DERA from Thermotoga maritima was found to be efficient for the aldol condensation and PDO production module. For the module of acetaldehyde supply from ethanol, an alcohol dehydrogenase from Hansenula polymorpha was selected. For the HCHO supply module, the control of HCHO concentration and its utilization were shown to be important for achieving the assimilation of HCHO in recombinant E.coli cells. By deleting the gene frmA for endogenous conversion of HCHO to formate and controlling HCHO at a level of about 0.6 mM, the concentration and yield of PDO were increased from initially 5.67 mM (0.43 g/L) and 0.057 mol/mol to 17.35 mM (1.32 g/L) and 0.096 mol/mol in bioconversion of ethanol and HCHO with resting E.coli cells. Further engineering of DERA and the HCHO supply module is necessary to realize the potential of this promising metabolic pathway.

DERA in Flow: Synthesis of a Statin Side Chain Precursor in Continuous Flow Employing Deoxyribose-5-Phosphate Aldolase Immobilized in Alginate-Luffa Matrix

Statins, cholesterol-lowering drugs used for the treatment of coronary artery disease (CAD), are among the top 10 prescribed drugs worldwide. However, the synthesis of their characteristic side chain containing two chiral hydroxyl groups can be challenging. The application of deoxyribose-5-phosphate aldolase (DERA) is currently one of the most promising routes for the synthesis of this side chain. Herein, we describe the development of a continuous flow process for the biosynthesis of a side chain precursor. Design of experiments (DoE) was used to optimize the reaction conditions (pH value and temperature) in batch. A pH of 7.5 and a temperature of 32.5 °C were identified to be the optimal process settings within the reaction space considered. Additionally, an immobilization method was developed using the alginate-luffa matrix (ALM), which is a fast, simple, and inexpensive method for enzyme immobilization. Furthermore, it is non-toxic, biodegradable, and from renewable resources. The final continuous process was operated stable for 4 h and can produce up to 4.5 g of product per day.

Engineering a short, aldolase-based pathway for (R)-1,3-butanediol production in Escherichia coli

Microbial processes can produce a wide range of compounds; however, producing complex and long chain hydrocarbons remains a challenge. Aldol condensation offers a direct route to synthesize these challenging chemistries and can be catalyzed by microbes using aldolases. Deoxyribose-5-phosphate aldolase (DERA) condenses aldehydes and/or ketones to β-hydroxyaldehydes, which can be further converted to value-added chemicals such as a precursor to cholesterol-lowering drugs. Here, we implement a short, aldolase-based pathway in Escherichia coli to produce (R)-1,3-BDO from glucose, an essential component of pharmaceutical products and cosmetics. First, we expressed a three step heterologous pathway from pyruvate to produce 0.3 g/L of (R)-1,3-BDO with a yield of 11.2 mg/g of glucose in wild-type E. coli K12 MG1655. We used a systems metabolic engineering approach to improve (R)-1,3-BDO titer and yield by: 1) identifying and reducing major by-products: ethanol, acetoin, and 2,3-butanediol; 2) increasing pathway flux through DERA to reduce accumulation of toxic acetaldehyde. We then implemented a two-stage fermentation process to improve (R)-1,3-BDO titer by 8-fold to 2.4 g/L and yield by 5-fold to 56 mg/g of glucose (11% of maximum theoretical yield) in strain BD24, by controlling pH to 7 and higher dissolved oxygen level. Furthermore, this study highlights the potential of the aldolase chemistry to synthesize diverse products directly from renewable resources in microbes.

1H, 13C, and 15N backbone and sidechain resonance assignments of a monomeric variant of E. coli deoxyribose-5-phosphate aldolase.

Deoxyribose-5-phosphate aldolase (DERA) catalyses the reversible conversion of 2-deoxyribose-5-phosphate (dR5P) into glyceraldehyde-3-phosphate (G3P) and acetaldehyde. For industrial applications, this enzyme is used in organic synthesis for aldol reactions between acetaldehyde as a donor and a wide range of aldehydes as acceptors. Here, we present a near complete set of sequence-specific 1H, 13C and 15N resonance assignments of a 28kDa monomeric variant of the Escherichia coli DERA. These assignments provide the basis for ongoing structural and dynamic analysis of DERA substrate specificity.

Accelerating Enzymatic Catalysis Using Vortex Fluidics.

Enzymes catalyze chemical transformations with outstanding stereo- and regio-specificities, but many enzymes are limited by their long reaction times. A general method to accelerate enzymes using pressure waves contained within thin films is described. Each enzyme responds best to specific frequencies of pressure waves, and an acceleration landscape for each protein is reported. A vortex fluidic device introduces pressure waves that drive increased rate constants (kcat ) and enzymatic efficiency (kcat /Km ). Four enzymes displayed an average seven-fold acceleration, with deoxyribose-5-phosphate aldolase (DERA) achieving an average 15-fold enhancement using this approach. In solving a common problem in enzyme catalysis, a powerful, generalizable tool for enzyme acceleration has been uncovered. This research provides new insights into previously uncontrolled factors affecting enzyme function.

Accelerating Enzymatic Catalysis Using Vortex Fluidics

AbstractEnzymes catalyze chemical transformations with outstanding stereo‐ and regio‐specificities, but many enzymes are limited by their long reaction times. A general method to accelerate enzymes using pressure waves contained within thin films is described. Each enzyme responds best to specific frequencies of pressure waves, and an acceleration landscape for each protein is reported. A vortex fluidic device introduces pressure waves that drive increased rate constants (kcat) and enzymatic efficiency (kcat/Km). Four enzymes displayed an average seven‐fold acceleration, with deoxyribose‐5‐phosphate aldolase (DERA) achieving an average 15‐fold enhancement using this approach. In solving a common problem in enzyme catalysis, a powerful, generalizable tool for enzyme acceleration has been uncovered. This research provides new insights into previously uncontrolled factors affecting enzyme function.

Engineered, highly productive biosynthesis of artificial, lactonized statin side-chain building blocks: The hidden potential of Escherichia coli unleashed

We have recently reported the development of an efficient, whole-cell process for chemoenzymatic production of key chiral intermediates in statin synthesis by employing high-density Escherichia coli culture with the overexpressed deoxyribose-5-phosphate aldolase (DERA). The optically pure, 6-substituted cyclic hemiacetals can be used for the synthesis of atorvastatin, rosuvastatin and pitavastatin using further chemical steps. All of the synthetic routes established to date begin with a regiospecific oxidation of these lactol intermediates into the corresponding lactones, followed by several steps yielding 6-substituted, open-chain or lactonized derivatives which can be coupled by various approaches with the heterocyclic part of the statin molecule. Here we report for the first time the use of PQQ-dependent glucose dehydrogenases for a highly efficient, regioselective oxidation of artificial, derivatized aldohexoses, more specifically, the statin lactol intermediates. First, PQQ-dependent dehydrogenases of both soluble and membrane-bound type were characterized for their activity toward various DERA-derived lactols. Further, we describe a highly productive whole-cell system for oxidation of these 2,4-dideoxyaldopyranoses using a PQQ-dependent glucose dehydrogenase (Gcd) overexpressed in E. coli while taking advantage of the respiratory chain as the mediator of the electron transfer to oxygen. Finally, a two-step artificial biosynthetic pathway was developed by unleashing the intrinsic genetic potential of E. coli. The combined overexpression of the endogenous DERA and the membrane-bound, PQQ-dependent glucose dehydrogenase, the latter being coupled to the respiratory chain, allows direct biosynthesis of 6-substituted lactones in a highly productive, high-yield, cost-effective and industrially scalable process.

Optimization of a Crude Deoxyribose-5-phosphate Aldolase Lyzate-Catalyzed Process in Synthesis of Statin Intermediates

A process for providing intermediate compounds as building blocks for effectively producing statins is described. The presented process is based on acetoxyacetaldehyde and acetaldehyde as substrates, which are presented in an aldol reaction catalyzed by a crude deoxyribose-5-phosphate aldolase (DERA) expressing culture lysate. Different addition regimes of both reactants into a reaction mixture were applied. For the highest concentration of product ((2S,4R)-4,6-dihydroxytetrahydro-2H-pyran-2-yl)methyl acetate, in the presented crude DERA expressing culture lysate-catalyzed reaction used further in the production of statins, the best addition time of reactants is described. Improved process conditions and reactants’ feeding regime were achieved by converting a batch reaction to a fed-batch process, reaching the highest concentration of product ((2S,4R)-4,6-dihydroxytetrahydro-2H-pyran-2-yl)methyl acetate near 77 g/L. The complete process was designed in a practical and economical manner and could be used further on an industrial scale.

Expression and Characterization of a Novel Deoxyribose 5-Phosphate Aldolase from Paenibacillus sp. EA001

A novel deoC gene was identified from Paenibacillus sp. EA001 isolated from soil. The gene had an open reading frame (ORF) of 663 base pairs encoding 220 amino acids with a molecular mass of 24.5 kDa. The amino acid sequence was 79 % identical to that of deoxyribose 5-phosphate aldolase (DERA) from Geobacillus sp. Y412MC10. The deoC gene encoding DERA was cloned into expression vector and the protein was expressed in Escherichia coli. The recombinant DERA was purified by using Ni-NTA affinity chromatography and characterized. The optimum temperature and pH for DERA were 50 degrees C and 6.0, respectively. The specific activity for deoxyribose 5-phosphate (DR5P), substrate, was 62 micronmol/min/mg. The Km value for DR5P was determined to be 145 mM with the Kcat value of 3.2 times 10(2 /sec) from Lineweaver-Burk plots. The EA001 DERA showed stability toward a high concentration of acetaldehyde (100 mM).

Cloning, expression, and characterization of a new deoxyribose 5-phosphate aldolase from Yersinia sp. EA015

A new deoC gene encoding deoxyribose 5-phosphate aldolase (DERA) was identified in Yersinia sp. EA015 isolated from soil. The DERA gene had an open reading frame (ORF) of 672 base pairs encoding 223 amino acids to yield a protein of molecular mass 24.8 kDa. The amino acid sequence was 94% identical to that of DERA from Yersinia intermedia ATCC 29909. DERA was over-expressed in Escherichia coli and purified using Ni–NTA affinity chromatography. The specific activity was 137 μmol/min/mg. The Michaelis constant ( k m value) of DERA was 9.1 mM. DERA was optimally active at pH 6.0 and 50 °C. DERA was tolerant to a high concentration (300 mM) of acetaldehyde.

The c-Myc Target Gene Rcl (C6orf108) Encodes a Novel Enzyme, Deoxynucleoside 5′-monophosphate N-Glycosidase

RCL is a c-Myc target with tumorigenic potential. Genome annotation predicted that RCL belonged to the N-deoxyribosyltransferase family. However, its putative relationship to this class of enzymes did not lead to its precise biochemical function. The purified native or N-terminal His-tagged recombinant rat RCL protein expressed in Escherichia coli exhibits the same enzyme activity, deoxynucleoside 5'-monophosphate N-glycosidase, never before described. dGMP appears to be the best substrate. RCL opens a new route in the nucleotide catabolic pathways by cleaving the N-glycosidic bond of deoxynucleoside 5'-monophosphates to yield two reaction products, deoxyribose 5-phosphate and purine or pyrimidine base. Biochemical studies show marked differences in the terms of the structure and catalytic mechanism between RCL and of its closest enzyme family neighbor, N-deoxyribosyltransferase. The reaction products of this novel enzyme activity have been implicated in purine or pyrimidine salvage, glycolysis, and angiogenesis, and hence are all highly relevant for tumorigenesis.

Development of an efficient, scalable, aldolase-catalyzed process for enantioselective synthesis of statin intermediates.

A process is reported for efficient, enantioselective production of key intermediates for the common chiral side chain of statin-type cholesterol-lowering drugs such as Lipitor (atorvastatin) and Crestor (rosuvastatin). The process features a one-pot tandem aldol reaction catalyzed by a deoxyribose-5-phosphate aldolase (DERA) to form a 6-carbon intermediate with installation of two stereogenic centers from 2-carbon starting materials. An improvement of almost 400-fold in volumetric productivity relative to the published enzymatic reaction conditions has been achieved, resulting in a commercially attractive process that has been run on up to a 100-g scale in a single batch at a rate of 30.6 g/liter per h. Catalyst load has been improved by 10-fold as well, from 20 to 2.0 wt % DERA. These improvements were achieved by a combination of discovery from environmental DNA of DERAs with improved activity and reaction optimization to overcome substrate inhibition. The two stereogenic centers are set by DERA with enantiomeric excess at >99.9% and diastereomeric excess at 96.6%. In addition, down-stream chemical steps have been developed to convert the enzymatic product efficiently to versatile intermediates applicable to preparation of atorvastatin and rosuvastatin.

Construction of chromosomally encoded secreted hemolysin fusion proteins by use of mini-Tn hlyA s transposon

We used the minitransposon Tn hlyA s [Gentschev, I., Maier, G., Kranig, A. and Goebel, W. (1996) Mol. Gen. Genet. 252, 266–274] for random insertion of the secretion signal (HlyA s) of Escherichia coli hemolysin (HlyA) into chromosomal genes. Four mini-Tn hlyA s derivatives bearing the gltA (citrate synthase), deoC (2 deoxyribose-5 phosphate aldolase), tig (trigger factor) genes and an unknown ORF fused to hlyA s were identified and characterized. Our data suggest that Tn hlyA s-generated hemolysin fusion proteins are secreted efficiently by the HlyB/HlyD/TolC hemolysin secretion machinery and that this can be useful for studies of gene expression or function.

Construction of chromosomally encoded secreted hemolysin fusion proteins by use of mini-TnhlyAs transposon.

We used the minitransposon TnhlyAs [Gentschev, I., Maier, G., Kranig, A. and Goebel, W. (1996) Mol. Gen. Genet. 252, 266-274] for random insertion of the secretion signal (HlyAs) of Escherichia coli hemolysin (HlyA) into chromosomal genes. Four mini-TnhlyAs derivatives bearing the gltA (citrate synthase), deoC (2 deoxyribose-5 phosphate aldolase), tig (trigger factor) genes and an unknown ORF fused to hlyAs were identified and characterized. Our data suggest that TnhlyAs-generated hemolysin fusion proteins are secreted efficiently by the HlyB/HlyD/TolC hemolysin secretion machinery and that this can be useful for studies of gene expression or function.

Deoxyribose 5-phosphate aldolase as a catalyst in asymmetric aldol condensation

Deoxyribose 5-phosphate aldolase as a catalyst in asymmetric aldol condensation

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.

Deoxyribose 5-phosphate aldolase: An enzyme that peaks in the G 2 phase of rat hepatoma cells

The activity of deoxyribose 5- P aldolase (2-deoxy- d-ribose 5-phosphate acetaldehyde lyase, EC 4.1.2.4) increases three- to fourfold in cultures of rat hepatoma cells that have reached stationary growth and have stopped in the G 2 phase of the cell cycle. We have shown that in cell cultures synchronized by three independent methods the enzyme activity peaks in late G 2, followed by a rapid decline during mitosis. Two compounds, dibutyryl cAMP and isoproterenol, that appear to block these cells in G 2, also cause a two- to fourfold increase in deoxyribose 5- P aldolase activity. It is suggested that this enzyme may play a role in lowering the pools of deoxyribonucleotides in the late G 2 M phase of the cell cycle.

An apparent lack of stereospecificity in the reaction catalysed by deoxyribose 5-phosphate aldolase due to methyl-group rotation and enolization before product release.

In the reaction catalysed by deoxyribose 5-phosphate aldolase (2-deoxy-D-ribose 5-phosphate acetaldehyde-lyase, EC 4.1.2.4) from Salmonella typhimurium, almost complete equilibration of the methyl-group protons of the product, acetaldehyde, occurs before its release from the enzyme surface. This phenomenon does not allow the stereo-chemical course of the reaction to be determined by using hydrogen-isotope labelling of the methyl group to generate a chiral centre.

Acrolein, an irreversible active-site-directed inhibitor of deoxyribose 5-phosphate aldolase?

The enzyme deoxyribose 5-phosphate aldolase was irreversibly inactivated by the substrate analogue acrolein with a pseudo-first-order rate constant of 0.324 min-1 and a Ki (apparent) of 2.7 x 10(-4) m. No inactivation was observed after prolonged incubation with the epoxide analogues glycidol phosphate and glycidaldehyde. It is suggested that the acrolein is first activated by forming a Schiff base with the enzyme active-site lysine residue and it is the activated inhibitor that reacts with a suitable-active-site nucleophile.