Alteration of the substrate specificity of benzoylformate decarboxylase from Pseudomonas putida by directed evolution

Alteration of the substrate specificity of thiamin diphosphate (ThDP)-dependent benzoylformate decarboxylase (BFD) by error-prone PCR is described. Two mutant enzymes, L476Q and M365L-L461S, were identified that accept ortho-substituted benzaldehyde derivatives as donor substrates, which leads to the formation of 2-hydroxy ketones. Both variants, L476Q and M365L-L461S, selectively catalyze the formation of enantiopure (S)-2-hydroxy-1-(2-methylphenyl)propan-1-one with excellent yields, a reaction which is only poorly catalyzed by the wild-type enzyme. Different ortho-substituted benzaldehyde derivatives, such as 2-chloro-, 2-methoxy-, or 2-bromobenzaldehyde are accepted as donor substrates by both BFD variants as well and conversion with acetaldehyde resulted in the corresponding (S)-2-hydroxy-1-phenylpropan-1-one derivatives. As deduced from modeling studies based on the 3D structure of wild-type BFD, reduction of the side chain size at position L461 probably results in an enlarged substrate binding site and facilitates the initial binding of ortho-substituted benzaldehyde derivatives to the cofactor ThDP.     

An activity, stability and selectivity comparison of propioin synthesis by thiamine diphosphate-dependent enzymes in a solid/gas bioreactor

Enzymatic carboligation in a solid/gas bioreactor represents a new challenge in biotechnology. In this paper, the continuous gas-phase production of propioin from two propanal molecules by using thiamine diphosphate-dependent enzymes was studied. Two enzymes were used, namely benzaldehyde lyase (BAL) from Pseudomonas fluorescens and benzoylformate decarboxylase (BFD) from Pseudomonas putida. The enzymes are homologous and catalyze carboligase and carbolyase reactions in which no external cofactor regeneration is needed. The influence of water and substrate activity on the initial reaction rate and biocatalyst stability was investigated. An increase in water activity raised the initial reaction rates to the maximal values of 250 and 80 U g(-1) for BAL and BFD, respectively. The half-life showed the same trend with maximal values of 50 and 78 min for BAL and BFD, respectively. The increase in the half-life by increasing water activity was unexpected. It was also observed that BFD is more stable than BAL in the presence of the substrate propanal. Both enzymes showed substrate inhibition in the kinetic studies, and BAL was also deactivated during the reaction. Unexpectedly, the stereoselectivity of both enzymes (ee of 19 % for BAL and racemic mixture for BFD) was significantly impaired in the gas phase compared to the liquid phase.     

α,β‐Unsaturated Aldehydes as Substrates for Asymmetric C?C Bond Forming Reactions with Thiamin Diphosphate (ThDP)‐Dependent Enzymes

The enzymes benzaldehyde lyase (BAL) from Pseudomonas fluorescens, benzoylformate decarboxylase (BFD) from Pseudomonas putida and pyruvate decarboxylase (PDC) from Saccharomyces cerevisiae provide different C C bond forming possibilities of α,β‐unsaturated aldehydes with aliphatic and aromatic aldehydes. Structure elucidation and determination of the absolute configuration of the products, which were obtained with high regio‐ and stereoselectivity were carried out. Selective 1,2‐reactivity with yields of 75% and >98% ee, for one single isomer ( A ) were obtained, by choosing the suitable enzyme in combination with the appropriate substrates. By varying enzymes or substrates the regioisomeric hydroxy ketones C , with up to >99% ee, can be obtained. The application of these new chiral building blocks in the synthesis of natural products or biological active substances is considerably facilitated by applying the different ThDP‐dependent enzymes as catalysts. Abbreviations: BAL, benzaldehyde lyase; BFD, benzoylformate decarboxylase; PDC, pyruvate decarboxylase; His, hexahistidine; 2‐HPP, 2‐hydroxy‐1‐phenylpropan‐1‐one; PAC, phenylacetylcarbinol; NTA, nitrilotriacetic acid; ThDP, thiamin diphosphate; wt, wild‐type.     

Inversion of stereoselectivity by applying mutants of the hydroxynitrile lyase from Manihot esculenta

The influence of Trp128-substituted mutants of the hydroxynitrile lyase from Manihot esculenta (MeHNL) on the stereoselectivity of MeHNL-catalyzed HCN additions to aldehydes with stereogenic centers, which yield the corresponding cyanohydrins, is described. In rac-2-phenylpropionaldehyde (rac-1) reactions, wild-type (wtMeHNL) and all MeHNL Trp128 mutants are highly (S)-selective toward the (R) enantiomer of rac-1; this results exclusively in (2S,3R)-cyanohydrin ((2S,3R)-2) with > or =96 % de. The (S) enantiomer of rac-1, however, only reacts (S)-selectively with wtMeHNL to give (2S,3S)-2 with 80 % de, whereas with Trp128 mutants, (R) selectivity increases with decreasing size of the amino acids exchanged. The MeHNL W128A mutant is exclusively (R)-selective, resulting in (2R,3S)-2 with 86 % de. The reaction behavior of rac-phenylbutyraldehyde (rac-5) is comparable with rac-1, which also inverts the stereoselectivity from (S) to (R) when the enzyme is exchanged from wtMeHNL to the W128A mutant. Stereogenic centers not adjacent to the aldehyde group, as in 7 and 9, do not influence the stereoselectivity of MeHNL catalysis, and (S) selectivity is observed in all cases. Stereoselectivity and inversion of stereoselectivity of MeHNL Trp128 mutant-catalyzed cyanohydrin formation can be explained and rationalized with crystal-structure-based molecular modeling.     

Enantioselective C?C Bond Ligation Using Recombinant Escherichia coli‐Whole‐Cell Biocatalysts

Thiamine diphosphate (ThDP)‐dependent enzymes like benzaldehyde lyase from Pseudomonas fluorescens (BAL) and benzoylformate decarboxylase from Pseudomonas putida (BFD) are versatile biocatalysts for the C C bond ligation of aldehydes to form enantiomerically pure 2‐hydroxy ketones. However, the large‐scale application of this enzyme class is often restricted by the required external addition of the expensive cofactor ThDP, as well as by the common use of dimethyl sulfoxide (DMSO) as a cosolvent, which leads to problems during the work‐up procedure. In the present paper we demonstrate that the addition of the excess cofactors, ThDP and magnesium ions (Mg²⁺), is not required when BAL or BFD are used in Escherichia coli resting cells. Furthermore, the combination of these resting cells with a biphasic reaction medium [methyl tert‐butyl ether (MTBE)/aqueous buffer] allows an increase of the substrate concentration up to 1 M, and an efficient extractive work‐up. As a practical example, e.g., the synthesis of (R)‐2‐hydroxy‐3,3‐dimethoxy‐phenylpropanone from benzaldehyde and 2,2‐dimethoxyacetaldehyde was optimized, achieving an isolated yield of 78 %, and an enantiomeric excess of 98 % ee in 24 h when operating at a substrate concentration of 0.4 M. The described reaction system in a biphasic medium is suitable for a wide range of aldehydes as substrates. The biphasic reaction medium minimizes also the formation of by‐products, which were observed when this reaction was performed in the conventional DMSO/buffer system.     

Molecular basis of lipase stereoselectivity

Lipases exhibit specific catalytic properties that make them attractive to biotechnological applications. Most important are the broad substrate specificity and the regio‐ and stereoselectivity of lipases. Despite mechanistic and structural similarities lipases differ significantly with respect to stereoselectivity toward natural and synthetic substrates. Models developed to describe and predict stereoselectivity toward certain types of synthetic substrates, e. g., secondary alcohols cannot be applied to natural acylglycerols, that are hydrolyzed by several animal and microbial lipases in a regioselective or stereoselective manner. Therefore, computer‐aided molecular modeling studies were used in order to predict the stereopreference of lipases toward triradylglycerols. Lipase variants with modified stereoselectivity properties toward triacylglycerols were engineered by re‐designing the recombinant enzyme. To understand the interactions governing lipase stereoselectivity towards natural substrates, knowledge of the structure of enzyme‐substrate complexes at the atomic level is essential. Such information can be obtained by X‐ray or NMR analysis of covalent enzyme‐inhibitor complexes. The crystal structures of enzymes complexed with triacylglycerol analog inhibitors allowed the identification of distinct binding sites for the three hydrophobic chains of the inhibitor.     

Factors mediating activity, selectivity, and substrate specificity for the thiamin diphosphate-dependent enzymes benzaldehyde lyase and benzoylformate decarboxylase

Benzaldehyde lyase from Pseudomonas fluorescens and benzoylformate decarboxylase from Pseudomonas putida are homologous thiamin diphosphate-dependent enzymes that catalyze carboligase and carbolyase reactions. Both enzymes catalyze the formation of chiral 2-hydroxy ketones from aldehydes. However, the reverse reaction has only been observed with benzaldehyde lyase. Whereas benzaldehyde lyase is strictly R specific, the stereoselectivity of benzoylformate decarboxylase from P. putida is dependent on the structure and orientation of the substrate aldehydes. In this study, the binding sites of both enzymes were investigated by using molecular modelling studies to explain the experimentally observed differences in the activity, stereo- and enantioselectivity and substrate specificity of both enzymes. We designed a detailed illustration that describes the shape of the binding site of both enzymes and sufficiently explains the experimental effects observed with the wild-type enzymes and different variants. These findings demonstrate that steric reasons are predominantly responsible for the differences observed in the (R)-benzoin cleavage and in the formation of chiral 2-hydroxy ketones.     

Stereoselective block of hERG channel by bupivacaine scrutinized at molecular level

In the heart, the hERG voltage-gated potassium channel mediates the I(Kr) current, which is crucial for the duration of cardiac action potential. Undesired block of the channel may prolong the QT interval with increased risk of malignant ventricular arrhythmia called torsades de pointes. Although the molecular determinants of hERG block are intensively studied, stereoselectivity has been poorly investigated. Levo-(S)-bupivacaine was the first drug reported to have higher affinity for hERG than its enantiomer. This study aims at understanding the principles underlying the stereoselectivity of bupivacaine block with the help of molecular modeling. Putative binding modes of levo-(S)- and dextro-(R)-bupivacaine inside an open form model of hERG channel were predicted by docking simulations, allowing a clear depiction of ligand-protein interactions. Estimated binding energies for both enantiomers to wild-type channel are in line with previously published electrophysiology measurements. These results may be considered as a confirmation at the molecular level of bupivacaine stereoselective binding towards hERG. Moreover this information lays the foundations for a structural guideline to filter out potentially cardiotoxic drug candidates in silico.     

Improving the carboligase activity of benzoylformate decarboxylase from Pseudomonas putida by a combination of directed evolution and site-directed mutagenesis

Benzoylformate decarboxylase (BFD) from Pseudomonas putida wassubjected to directed molecular evolution to generate mutantswith increased carboligase activity which is a side reactionof the enzyme. After a single round of random mutagenesis mutantswere isolated which exhibited a 5-fold increased carboligaseactivity in aqueous buffer compared to the wild-type enzymewith a high enantiomeric excess of the product (S)-2-hydroxy-1-phenyl-propanone.From the same library, mutants with enhanced carboligase activityin water-miscible organic solvents have been isolated. The selectedmutants have been characterized by sequencing, revealing thatall mutants carry a mutation at Leu476, which is close to theactive site but does not directly interact with the active center.BFD-L476Q has a 5-fold higher carboligase activity than thewild-type enzyme. L476 was subjected to saturation mutagenesisyielding eight different mutants with up to 5-fold increasedcarboligase activity. Surprisingly, all L476 mutants catalyzethe formation of 2-hydroxy-1-phenyl-propanone with significantlyhigher enantioselectivity than the wild-type enzyme althoughenantioselectivity was not a selection parameter. Leu476 potentiallyplays the role of a gatekeeper of the active site of BFD, possiblyby controlling the release of the product. The biocatalyst couldbe significantly improved for its side reaction, the C–Cbond formation and for application under conditions that arenot optimized in nature.     

Stereospecific Reduction of Methyl o‐Chlorobenzoylformate at 300 g⋅L−1 without Additional Cofactor using a Carbonyl Reductase Mined from Candida glabrata

In order to search for oxidoreductases suitable for the preparation of methyl (R)‐o‐chloromandelate [(R)‐CMM], the key intermediate for clopidogrel, the homologous proteins of Gre2p were expressed in Escherichia coli, among which CgKR1 showed the most satisfactory activity and stereoselectivity towards methyl o‐chlorobenzoylformate (CBFM). Using the crude enzyme of CgKR1 and glucose dehydrogenase (GDH), as much as 300 g⋅L⁻¹ of CBFM was almost stoichiometrically converted to (R)‐CMM with excellent enantiomeric excess (98.7% ee). More importantly, the reaction could be performed without external addition of an expensive cofactor. The substrate profile indicates that keto esters serve as the most suitable substrate, which was confirmed by gram‐scale preparations. Homology modeling and docking analysis revealed the molecular basis for the high stereoselectivity of CgKR1. These demonstrate not only the feasibility of in silico mining of novel enzymes based on sequence homology but also the applicability of this new reductase for the practical production of optically active (R)‐CMM.     

Rational design of Rhizopus oryzae lipase with modified stereoselectivity toward triradylglycerols

The binding site of sn-1(3)-regioselective Rhizopus oryzae lipase (ROL) has been engineered to change the stereoselectivity of hydrolysis of triacylglycerol substrates and analogs. Two types of prochiral triradylglycerols were considered: 'flexible' substrates with ether, benzylether or ester groups, and 'rigid' substrates with amide or phenyl groups, respectively, in the sn-2 position. The molecular basis of sn-1(3) stereoselectivity of ROL was investigated by modeling the interactions between substrates and ROL, and the model was confirmed by experimental determination of the stereoselectivity of wild-type and mutated ROL. For the substrates, the following rules were derived: (i) stereopreference of ROL toward triradylglycerols depends on the substrate structure. Substrates with 'flexible' sn-2 substituents are preferably hydrolyzed at sn-1, 'rigid' substrates at sn-3. (ii) Stereopreference of ROL toward triradylglycerols can be predicted by analyzing the geometry of the substrate docked to ROL: if the torsion angle phiO3-C3 of glycerol is more than 150 degrees, the substrate will preferably be hydrolyzed in sn-1, otherwise in sn-3. For ROL, the following rules were derived: (i) residue 258 affects stereoselectivity by steric interactions with the sn-2 substituent rather than polar interactions. To a lower extent, stereoselectivity is influenced by mutations further apart (L254) from residue 258. (ii) With 'rigid' substrates, increasing the size of the binding site (mutations L258A and L258S) shifts stereoselectivity of hydrolysis toward sn-1, decreasing its size (L258F and L258F/L254F) toward sn-3.      

Construction of Mutant Glucose Oxidases with Increased Dye-Mediated Dehydrogenase Activity

Mutagenesis studies on glucose oxidases (GOxs) were conducted to construct GOxs with reduced oxidase activity and increased dehydrogenase activity. We focused on two representative GOxs, of which crystal structures have already been reported—Penicillium amagasakiense GOx (PDB ID; 1gpe) and Aspergillus niger GOx (PDB ID; 1cf3). We constructed oxygen-interacting structural models for GOxs, and predicted the residues responsible for oxidative half reaction with oxygen on the basis of the crystal structure of cholesterol oxidase as well as on the fact that both enzymes are members of the glucose/methanol/choline (GMC) oxidoreductase family. Rational amino acid substitution resulted in the construction of an engineered GOx with drastically decreased oxidase activity and increased dehydrogenase activity, which was higher than that of the wild-type enzyme. As a result, the dehydrogenase/oxidase ratio of the engineered enzyme was more than 11-fold greater than that of the wild-type enzyme. These results indicate that alteration of the dehydrogenase/oxidase activity ratio of GOxs is possible by introducing a mutation into the putative functional residues responsible for oxidative half reaction with oxygen of these enzymes, resulting in a further increased dehydrogenase activity. This is the first study reporting the alteration of GOx electron acceptor preference from oxygen to an artificial electron acceptor.     

苯甲醛直接氯化法合成间氯苯甲醛

以苯甲醛为原料直接氯化合成间氯苯甲醛,通过对几种可能的催化剂进行比较,选择了无水三氯化铝作为该氯化反应的催化剂;用单因素方法和正交实验考察了原料液浓度、催化剂用量、反应温度和反应时间对氯化反应的影响,得到了氯化反应优惠条件:苯甲醛1 33mol/L,无水三氯化铝1 70mol/L,反应时间30min,反应温度50℃。苯甲醛的转化率≥90%,间氯苯甲醛的收率≥79%,选择性为88%,氯化产品中基本无同分异构体。     

Identification and Biosynthesis of New Acyloins from the Thermophilic Bacterium Thermosporothrix hazakensis SK20‐1T

Two new acyloin compounds were isolated from the thermophilic bacterium Thermosporothrix hazakensis SK20‐1^T. Genome sequencing of the bacterium and biochemical studies identified the thiamine diphosphate (TPP)‐dependent enzyme Thzk0150, which is involved in the formation of acyloin. Through extensive analysis of the Thzk0150‐catalyzed reaction products, we propose a putative reaction mechanism involving two substrates: 4‐methyl‐2‐oxovalerate as an acyl donor and phenyl pyruvate as an acyl acceptor.     

Exchanging the substrate specificities of pyruvate decarboxylase from Zymomonas mobilis and benzoylformate decarboxylase from Pseudomonas putida

Pyruvate decarboxylase from Zymomonas mobilis (PDC) and benzoylformate decarboxylase from Pseudomonas putida (BFD) are thiamine diphosphate-dependent enzymes that decarboxylate 2-keto acids. Although they share a common homotetrameric structure they have relatively low sequence similarity and different substrate spectra. PDC prefers short aliphatic substrates whereas BFD favours aromatic 2-keto acids. These preferences are also reflected in their carboligation reactions. PDC catalyses the conversion of benzaldehyde and acetaldehyde to (R)-phenylacetylcarbinol and predominantly (S)-acetoin, whereas (R)-benzoin and mainly (S)-2-hydroxypropiophenone are the products of BFD catalysis. Comparison of the X-ray structures of both enzymes identified two residues in each that were likely to be involved in determining substrate specificity. Site-directed mutagenesis was used to interchange these residues in both BFD and PDC. The substrate range and kinetic parameters for the decarboxylation reaction were studied for each variant. The most successful variants, PDCI472A and BFDA460I, catalysed the decarboxylation of benzoylformate and pyruvate, respectively, although both variants now preferred the long-chain aliphatic substrates, 2-ketopentanoic and 2-ketohexanoic acid. With respect to the carboligase activity, PDCI472A proved to be a real chimera between PDC and BFD whereas BFDA460I/F464I provided the most interesting result with an almost complete reversal of the stereochemistry of its 2-hydroxypropiophenone product.     

Molecular modeling and experimental verification of lipase-catalyzed enantioselective esterification of racemic naproxen in supercritical carbon dioxide

Experimental and simulation analyses were performed on the lipase-catalyzed esterification reaction of racemic naproxen by CALB (candida antarctica lipase B) enzyme in supercritical carbon dioxide. The reaction pathways were investigated by quantum mechanical analysis, and the enantioselectivity of the products was predicted by molecular dynamics simulation analysis. Calculated results from molecular modeling in supercritical carbon dioxide were qualitatively compared with experimental data by using racemic naproxen as a substrate. All molecular modeling results and experimental data were acquired and compared with those in ambient and supercritical condition. Moreover, to verify the stability of enzymatic reaction in each solvent condition, reaction pathways were investigated in several solvent conditions (vacuum, water, hexane and supercritical carbon dioxide), and the stability of enzymatic reaction in supercritical carbon dioxide was compared with other solvent conditions. This paper is dedicated to Professor Chul Soo Lee on the occasion of his retirement from Korea University.     

Enantioselective Hosts for Neutral Derivatives of Amino Acids

A series of macropolycyclic receptors for hydrogen bond donor/acceptor guests were prepared and studied. Molecular modeling and X-ray crystallography provided structural information, while NMR titration studies allowed determination of free energies of association. Some of die receptors prepared showed enantioselectivity as high as 3 kcal/mol in binding derivatives of simple amino acids in organic solvents. These studies show that conformational homogeneity is one of the most important characteristics of a selective host and that molecular modeling can play an important role in the design of such substances.     

Lipase-catalyzed synthesis of chiral triglycerides

Under certain reaction conditions, the acidolysis of tripalmitin with oleic acid using immobilized lipase from Rhizomucor miehei resulted in a higher level of monosubstituted oleoyldipalmitoyl (OPP) triglycerides than had been predicted according to kinetic modeling. The reaction products were subjected to chiral analysis by high-performance liquid chromatography (HPLC), which indicated that the enzyme was more active at the sn-1 position of the triglyceride than at the sn-3 position, resulting in synthesis of the chiral triglyceride 1-oleoyl-2,3-dipalmitoyl-sn-glycerol. A kinetic model was developed and was correlated with the HPLC method to provide a simple means to predict the stereoselectivity of lipase-catalyzed reactions. By using the model, the stereoselectivity of immobilized Rhizomucor miehei lipase was found to depend strongly on the initial water activity (a _w) of the reaction mixture, with greater selectivity occurring at lower a _w. The sn-1 selectivity was essentially maintained using various solvents, or without solvent, when a _w was kept constantly low. Variation in the fatty acid composition of the triglyceride indicated that shorter-chain fatty acids result in greater stereoselectivity, while variation of the chainlength of the free fatty acid indicated an enhancement by the longest chainlength. The stereoselectivity of this lipase was confirmed using a new ¹³C nuclear magnetic resonance method. By using immobilized R. miehei lipase at low a _w approximately 80% of the chiral triglyceride found in the reaction mixture was the sn-1 enantiomer, at high reaction conversion.     

Scalaradial, a dialdehyde-containing marine metabolite that causes an unexpected noncovalent PLA2 Inactivation

Several marine terpenoids that contain at least one reactive aldehyde group, such as manoalide and its congeners, possess interesting anti-inflammatory activities that are mediated by the covalent inactivation of secretory phospholipase A(2) (sPLA(2)). Scalaradial, a 1,4-dialdehyde marine terpenoid that was isolated from the sponge Cacospongia mollior, is endowed with a relevant anti-inflammatory profile, both in vitro and in vivo, through selective sPLA(2) inhibition. Due to its peculiar dialdehyde structural feature, it has been proposed that scalaradial exerts its enzymatic inactivation by means of an irreversible covalent modification of its target. In the context of our on-going research on anti-PLA(2) natural products and their interaction at a molecular level, we studied scalaradial in an attempt to shed more light on the molecular mechanism of its PLA(2) inhibition. A detailed analysis of the reaction profile between scalaradial and bee venom PLA(2), a model sPLA(2) that shares a high structural homology with the human synovial enzyme, was performed by a combination of spectroscopic techniques, chemical reactions (selective modifications, biomimetic reactions), and classical protein chemistry (such as proteolytic digestion, HPLC and mass spectrometry), along with molecular modeling studies. Unexpectedly, our data clearly indicated the noncovalent forces to be the leading event in the PLA(2) inactivation process; thus, the covalent modification of the enzyme emerges as only a minor side event in the ligand-enzyme interaction. The overall picture might be useful in the design of SLD analogues as new potential anti-inflammatory compounds that target sPLA(2) enzymes.     

1–氨基四唑的三步法合成与表征

研究了以苯甲醛、水合肼为原料,经缩合、成环和水解三步反应合成1–氨基四唑的方法,采用红外光谱、核磁共振、元素分析等对中间体及产物结构进行了表征。考察了缩合反应温度、成环反应中原甲酸三乙酯与苯甲醛腙的摩尔比对反应收率的影响,确定了最佳反应条件:缩合反应温度为0⁵℃,n(原甲酸三乙酯)∶n(苯甲醛腙)=1.8∶1。三步反应总收率为49.5%。