Tools and ingredients for the biocatalytic synthesis of carbohydrates and glycoconjugates

The presence of multiple functional groups and stereocentres in carbohydrates and glycoconjugates make them challenging targets for synthesis. Although progress in chemical synthesis and engineering is impressive, there is still a need to selectively introduce and remove protecting groups in the total synthesis of target molecules of increasing complexity. Multiple hydroxyl-groups with similar reactivities have to be differentiated in order to form the desired glycosidic bonds in a regio- and stereospecific way. To complement the existing chemical tools and ingredients, biocatalysts for selective carbon–carbon bond formation and glycosylation reactions have been developed. The availability of auxiliary ingredients like transfer reagents is a prerequisite for the development of viable biocatalytic process steps. In the case of dihydroxyacetone-phosphate-dependent aldolases, e.g. fructose-1,6-bisphosphate aldolase (EC 4.1.2.13), the large-scale availability of dihydroxyacetone-phosphate (DHAP) eliminates the need to synthesize the donor DHAP. For the pyruvate-dependent aldolases, e.g. the N-acetylneuraminic acid aldolase (EC 4.1.3.3) and acetaldehyde-dependent aldolases like the 2-deoxy-d-ribose-5-phosphate aldolase (4.2.1.4), the donors pyruvate and acetaldehyde are also available on a large scale. A broad range of natural and recombinant aldolases have been produced in stable lyophilized form. Recombinant transketolase together with a new synthesis of hydroxypyruvates has provided a platform technology for the preparation of monosaccharides, whereby the carbon backbone is extended by a two-carbon unit (C2-elongation). Natural and recombinant glycosyltransferases have been prepared on a large-scale to establish biocatalytic glycosylations in water as highly regio- and stereospecific reaction methodologies without the need for laborious protecting group manipulations, solubility adaptations and complex synthetic schemes. In order to simplify the synthetic manipulations for specific glycosylations, toolkits for β-1,4-galactosylations, α-1,3-galactosylations and α-1,3-fucosylations have been developed for rapid quantitative conversions. The introduction of matched pairs of biocatalysts and transfer reagents as ingredients together with the optimized reaction methodology as tool provide an important starting point for biocatalytic glycomics.     

Localization of fetal aldolases during early stages of azo-dye hepatocarcinogenesis in rat

We have looked for the synthesis of fetal aldolases A and C during the early stages of hepatocarcinogenesis induced by 3′-methyl-4-(dimethylamino) azobenzene in the rat. Using indirect immunoperoxidase and immunofluorescence techniques we show that oval and transitional cells are the main cellular sites of fetal aldolases A and C production while hepatocytes only synthesize aldolase B. The synthesis of aldolases A and C was confirmed by electrophoresis analysis. These results indicate that different cell types are involved in fetal aldolase production during the early stages of azo-dye feeding and during regeneration after carbon tetrachloride intoxication where the synthesis of these isozymes is restricted to sinusoïdal cells.     

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.     

Two Distinct Aldolases of Class II Type in the Cyanoplasts and in the Cytosol of the Alga Cyanophora paradoxa

Two aldolases from the alga Cyanophora paradoxa (Glaucocystophyta) can be separated by chromatography on diethylaminoethyl-Fractogel. The two aldolases are inhibited by 1 mM ethylene-diaminetetraacetate (EDTA) and, therefore, are class II aldolases. When cells of C. paradoxa were fractionated, one aldolase was associated with the cytosol fraction and the other was associated with the cyanoplast fraction. The Km(fructose-1,6-bisphosphate) was 600 [mu]M for the cytosolic aldolase and 340 [mu]M for the cyanoplast aldolase. The activity of the cytosolic aldolase was increased up to 4-fold by 100 mM K+ and slightly inhibited by Li+ and Cs+, whereas the cyanoplast aldolase was not affected by these ions. Inactivation by 1 mM EDTA could be partly restored by the addition of Co2+ or Mn2+ and to a lesser extent by Zn2+ or Mg2+. The molecular masses of the native cytosolic and cyanoplast aldolases are about 90 and 85 kD, respectively, as estimated by velocity centrifugation in sucrose gradients. Implications for the evolution of class I and II aldolases in chloroplasts of higher plants and algae will be discussed.     

Chemical and enzymatic routes to dihydroxyacetone phosphate

Stereoselective carbon–carbon bond formation with aldolases has become an indispensable tool in preparative synthetic chemistry. In particular, the dihydroxyacetone phosphate (DHAP)-dependent aldolases are attractive because four different types are available that allow access to a complete set of diastereomers of vicinal diols from achiral aldehyde acceptors and the DHAP donor substrate. While the substrate specificity for the acceptor is rather relaxed, these enzymes show only very limited tolerance for substituting the donor. Therefore, access to DHAP is instrumental for the preparative exploitation of these enzymes, and several routes for its synthesis have become available. DHAP is unstable, so chemical synthetic routes have concentrated on producing a storable precursor that can easily be converted to DHAP immediately before its use. Enzymatic routes have concentrated on integrating the DHAP formation with upstream or downstream catalytic steps, leading to multi-enzyme arrangements with up to seven enzymes operating simultaneously. While the various chemical routes suffer from either low yields, complicated work-up, or toxic reagents or catalysts, the enzymatic routes suffer from complex product mixtures and the need to assemble multiple enzymes into one reaction scheme. Both types of routes will require further improvement to serve as a basis for a scalable route to DHAP.     

Microbial aldolases as C–C bonding enzymes—unknown treasures and new developments

Aldolases are a specific group of lyases that catalyze the reversible stereoselective addition of a donor compound (nucleophile) onto an acceptor compound (electrophile). Whereas most aldolases are specific for their donor compound in the aldolization reaction, they often tolerate a wide range of aldehydes as acceptor compounds. C–C bonding by aldolases creates stereocenters in the resulting aldol products. This makes aldolases interesting tools for asymmetric syntheses of rare sugars or sugar-derived compounds as iminocyclitols, statins, epothilones, and sialic acids. Besides the well-known fructose 1,6-bisphosphate aldolase, other aldolases of microbial origin have attracted the interest of synthetic bio-organic chemists in recent years. These are either other dihydroxyacetone phosphate aldolases or aldolases depending on pyruvate/phosphoenolpyruvate, glycine, or acetaldehyde as donor substrate. Recently, an aldolase that accepts dihydroxyacetone or hydroxyacetone as a donor was described. A further enlargement of the arsenal of available chemoenzymatic tools can be achieved through screening for novel aldolase activities and directed evolution of existing aldolases to alter their substrate- or stereospecifities. We give an update of work on aldolases, with an emphasis on microbial aldolases.     

两种新型L-苏氨酸醛缩酶的鉴定及活性检测方法

为了实现重要医药中间体p-羟基-α-氨基酸的生物酶法合成,挖掘验证新型的L-苏氨酸醛缩酶.以pET-28a(+)作为表达载体,通过蛋白表达纯化、薄层层析色谱(TLC)和高效液相色谱(HPLC)技术分析L-苏氨酸醛缩酶及其催化产物的性质.基于4-氨基-3-肼基-5-巯基-1,2,4-三氮唑(Purpald)显色试剂开发检...     

Directed evolution of aldolases for exploitation in synthetic organic chemistry

This review focuses on the directed evolution of aldolases with synthetically useful properties. Directed evolution has been used to address a number of limitations associated with the use of wild-type aldolases as catalysts in synthetic organic chemistry. The generation of aldolase enzymes with a modified or expanded substrate repertoire is described. Particular emphasis is placed on the directed evolution of aldolases with modified stereochemical properties: such enzymes can be useful catalysts in the stereoselective synthesis of biologically active small molecules. The review also describes some of the fundamental insights into mechanistic enzymology that directed evolution can provide.     

In vivo selection for the directed evolution of L-rhamnulose aldolase from L-rhamnulose-1-phosphate aldolase (RhaD)

Dihydroxyacetone phosphate (DHAP)-dependent aldolases have been widely used for organic synthesis. The major drawback of DHAP-dependent aldolases is their strict donor substrate specificity toward DHAP, which is expensive and unstable. Here we report the development of an in vivo selection system for the directed evolution of the DHAP-dependent aldolase, L-rhamnulose-1-phosphate aldolase (RhaD), to alter its donor substrate specificity from DHAP to dihydroxyacetone (DHA). We also report preliminary results on mutants that were discovered with this screen. A strain deficient in the L-rhamnose metabolic pathway in Escherichia coli (DeltarhaDAB, DE3) was constructed and used as a selection host strain. Co-expression of L-rhamnose isomerase (rhaA) and rhaD in the selection host did not restore its growth on minimal plate supplemented with L-rhamnose as a sole carbon source, because of the lack of L-rhamnulose kinase (RhaB) activity and the inability of WT RhaD aldolase to use unphosphorylated L-rhamnulose as a substrate. Use of this selection host and co-expression vector system gives us an in vivo selection for the desired mutant RhaD which can cleave unphosphorylated L-rhamnulose and allow the mutant to grow in the minimal media. An error-prone PCR (ep-PCR) library of rhaD gene on the co-expression vector was constructed and introduced into the rha-mutant, and survivors were selected in minimal media with l-rhamnose (MMRha media). An initial round of screening gave mutants allowing the selection strain to grow on MMRha plates. This in vivo selection system allows rapid screening of mutated aldolases that can utilize dihydroxyacetone as a donor substrate.     

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.     

Surface Free Energy‐Induced Assembly to the Synthesis of Grid‐Like Multicavity Carbon Spheres with High Level In‐Cavity Encapsulation for Lithium–Sulfur Cathode

Carbon microcapsules with a large interior cavity and porous shell are ideal hosts for guest species, while to maximize in‐cavity volume has always been a challenge. Herein, a surface free energy‐induced assembly approach is proposed for synthesis of multicavity carbon spheres (MCC). When used as a host for lithium–sulfur cathodes, MCC are fully accessible for sulfur—with high level in‐cavity encapsulation ability of grid‐like cavities. The crucial point for this assembly approach is the employment of small sized nanoemulsions with high homogeneity as primary building blocks. Spontaneous aggregation and assembly of substructural units are processing in following hydrothermal synthesis induced by reduction of surface free energy of system. As a result, multicavity structure is formed, where the size and number of cavities can be modulated by changing size of nanoemulsion and concentration of polymer. Confined pyrolysis enables to further enlarge cavity size compared to regular pyrolysis. The carbon–sulfur cathode exhibits excellent cycling stability and rate performance, i.e., high capacity of 943 and 869 mA h g^?1 after 200 cycles at current density of 0.5 and 2.0 C. The strategy has paved the way for custom‐ordered synthesis of nanostructured carbon with keen demands in high loading capacity of guest species.     

Characterization of chloroplastic fructose 1,6-bisphosphate aldolases as lysine-methylated proteins in plants

In pea (Pisum sativum), the protein-lysine methyltransferase (PsLSMT) catalyzes the trimethylation of Lys-14 in the large subunit (LS) of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the enzyme catalyzing the CO(2) fixation step during photosynthesis. Homologs of PsLSMT, herein referred to as LSMT-like enzymes, are found in all plant genomes, but methylation of LS Rubisco is not universal in the plant kingdom, suggesting a species-specific protein substrate specificity of the methyltransferase. In this study, we report the biochemical characterization of the LSMT-like enzyme from Arabidopsis thaliana (AtLSMT-L), with a focus on its substrate specificity. We show that, in Arabidopsis, LS Rubisco is not naturally methylated and that the physiological substrates of AtLSMT-L are chloroplastic fructose 1,6-bisphosphate aldolase isoforms. These enzymes, which are involved in the assimilation of CO(2) through the Calvin cycle and in chloroplastic glycolysis, are trimethylated at a conserved lysyl residue located close to the C terminus. Both AtLSMT-L and PsLSMT are able to methylate aldolases with similar kinetic parameters and product specificity. Thus, the divergent substrate specificity of LSMT-like enzymes from pea and Arabidopsis concerns only Rubisco. AtLSMT-L is able to interact with unmethylated Rubisco, but the complex is catalytically unproductive. Trimethylation does not modify the kinetic properties and tetrameric organization of aldolases in vitro. The identification of aldolases as methyl proteins in Arabidopsis and other species like pea suggests a role of protein lysine methylation in carbon metabolism in chloroplasts.     

Synthesis of beta-hydroxy-alpha-amino acids with a reengineered alanine racemase

The Y265A mutant of alanine racemase (alrY265A) was evaluated as a catalyst for the synthesis of beta-hydroxy-alpha-amino acids. It promotes the PLP-dependent aldol condensation of glycine with a range of aromatic aldehydes. The desired products were obtained with complete stereocontrol at C(alpha) (ee>99%, D) and moderate to high selectivity at C(beta) (up to 97% de). The designed enzyme is thus similar to natural d-threonine aldolases in its substrate specificity and stereoselectivity. Moreover, its ability to utilize alanine as an alternative donor suggests an expanded scope of potential utility for the production of biologically active compounds.     

Enzymatic biocatalysis in chemical synthesis of pharmaceuticals (Review)

In this review we summarize the available research on enzymatic biocatalysis in the chemical synthesis of drugs. We focus on oxydoreductsases, particularly ketoreductases, that are widely used in biotechnological processes: alpha- and omega-transaminases, lipases, nitrile hydrolases, and aldolases. The potential for the extended use of novel enzymes produced via bioengineering is discussed.     

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.     

Post Iron Decoration of Mesoporous Nitrogen‐Doped Carbon Spheres for Efficient Electrochemical Oxygen Reduction

Iron–nitrogen–carbon (Fe–N–C) catalysts are considered as the most promising nonprecious metal catalysts for oxygen reduction reactions (ORRs). Their synthesis generally involves complex pyrolysis reactions at high temperature, making it difficult to optimize their composition, pore structure, and active sites. This study reports a simple synthesis strategy by reacting preformed nitrogen‐doped carbon scaffolds with iron pentacarbonyl, a liquid precursor that can effectively form active sites with the nitrogen sites, enabling more effective control of the catalyst. The resultant catalyst possesses a well‐defined mesoporous structure, a high surface area, and optimized active sites. The catalysts exhibit high ORR activity comparable to that of Pt/C catalyst (40% Pt loading) in alkaline media, with excellent stability and methanol tolerance. The synthetic strategy can be extended to synthesize other metal–N–C catalysts.     

Stereoselectivity of fructose-1,6-bisphosphate aldolase in Thermus caldophilus

It was recently established that fructose-1,6-bisphosphate (FBP) aldolase (FBA) and tagatose-1,6-bisphosphate (TBP) aldolase (TBA), two class II aldolases, are highly specific for the diastereoselective synthesis of FBP and TBP from glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP), respectively. In this paper, we report on a FBA from the thermophile Thermus caldophilus GK24 (Tca) that produces both FBP and TBP from C(3) substrates. Moreover, the FBP:TBP ratio could be adjusted by manipulating the concentrations of G3P and DHAP. This is the first native FBA known to show dual diastereoselectivity among the FBAs and TBAs characterized thus far. To explain the behavior of this enzyme, the X-ray crystal structure of the Tca FBA in complex with DHAP was determined at 2.2A resolution. It appears that as a result of alteration of five G3P binding residues, the substrate binding cavity of Tca FBA has a greater volume than those in the Escherichia coli FBA-phosphoglycolohydroxamate (PGH) and TBA-PGH complexes. We suggest that this steric difference underlies the difference in the diastereoselectivities of these class II aldolases.     

Characterization, cloning, and evolutionary history of the chloroplast and cytosolic class I aldolases of the red alga Galdieria sulphuraria

Two fructose-1,6-bisphosphate aldolases from the acido- and thermophilic red alga Galdieria sulphuraria were purified to apparent homogeneity and N-terminally microsequenced. Both aldolases had similar biochemical properties such as Km (FBP) (5.6-5.8 microM) and molecular masses of the native enzymes (165kDa) as determined by size exclusion chromatography. The subunit size of the purified aldolases, as determined by SDS-PAGE, was 42kDa for both aldolases. The isoenzymes were not inhibited by EDTA or affected by cysteine or potassium ions, implying that they belong to the class I group of aldolases, while other red algae are known to have one class I and one class II aldolase inhibited by EDTA. cDNA clones of the cytosolic and plastidic aldolases were isolated and sequenced. The gene for the cytosolic isoenzyme contained a 303bp untranslated leader sequence, while the gene for the plastidic isoenzyme exhibited a transit sequence of 56 amino-acid residues. Both isoenzymes showed about 48% homology in the deduced amino-acid sequences. A gene tree relates both aldolases to the basis of early eukaryotic class I aldolases. The phylogenetic relationship to other aldolases, particularly to cyanobacterial class II aldolases, is discussed.