Active Site Mapping of Human Cathepsin F with Dipeptide Nitrile Inhibitors

Cleavage of the invariant chain is the key event in the trafficking pathway of major histocompatibility complex class II. Cathepsin S is the major processing enzyme of the invariant chain, but cathepsin F acts in macrophages as its functional synergist which is as potent as cathepsin S in invariant chain cleavage. Dedicated low‐molecular‐weight inhibitors for cathepsin F have not yet been developed. An active site mapping with 52 dipeptide nitriles, reacting as covalent–reversible inhibitors, was performed to draw structure–activity relationships for the non‐primed binding region of human cathepsin F. In a stepwise process, new compounds with optimized fragment combinations were designed and synthesized. These dipeptide nitriles were evaluated on human cysteine cathepsins F, B, L, K and S. Compounds 10 (N‐(4‐phenylbenzoyl)‐leucylglycine nitrile) and 12 (N‐(4‐phenylbenzoyl)leucylmethionine nitrile) were found to be potent inhibitors of human cathepsin F, with K_i values <10 nM . With all dipeptide nitriles from our study, a 3D activity landscape was generated to visualize structure–activity relationships for this series of cathepsin F inhibitors.     

Nocardia sp.腈水合酶的纯化过程研究

对Nocardiasp.高活力腈水合酶进行了纯化研究。在细胞破碎中,超声时间对腈水合酶比酶活存在一个最优值,超声时间为20.00min时得到的比酶活最高。在离子交换层析过程中,采用DEAE-Sepharose作为层析介质,分别对平衡缓冲液pH、离子强度和线性梯度洗脱体积进行了优化。结果表明,采用pH7.20、50mmolL-1的Na2HPO4-NaH2PO4溶液作为平衡缓冲液,0.00~1.00molL-1的NaCl线性梯度洗脱,洗脱体积为20~25倍柱体积,此条件下腈水合酶的纯化倍数和酶活收率较佳。以Phenyl-SepharoseFF为层析介质研究了疏水层析精制腈水合酶的工艺过程,采用两步层析优化方法纯化出的Nocardiasp.腈水合酶比酶活达到2648.0U穖g-1,酶活收率为40.84%,用SDS-PAGE检测其纯度为99.00%以上。Nocardiasp.腈水合酶的两个亚基分子量分别为22.90kDa和27.38kDa。     

Nitrile Hydratase Activity of a Recombinant Nitrilase

Appreciable amounts of amide are formed in the course of nitrile hydrolysis in the presence of recombinant nitrilase from Pseudomonas fluorescens EBC 191, depending on the α‐substituent and the reaction conditions. The ratio of the nitrilase and nitrile hydratase activities of the enzyme is profoundly influenced by the electronic and steric properties of the reactant. In general, amide formation increased when the α‐substituent was electron‐deficient; 2‐chloro‐2‐phenylacetonitrile, for example, afforded 89 % amide. We found, moreover, that (R)‐mandelonitrile was hydrolysed with 11 % of amide formation whereas 55 % amide was formed from the (S)‐enantiomer; a similar effect was found for the O‐acetyl derivatives. A mechanism that accomodates our results is proposed.     

Biocatalytic Synthesis of Nitriles through Dehydration of Aldoximes: The Substrate Scope of Aldoxime Dehydratases

Nitriles, which are mostly needed and produced by the chemical industry, play a major role in various industry segments, ranging from high‐volume, low‐price sectors, such as polymers, to low‐volume, high‐price sectors, such as chiral pharma drugs. A common industrial technology for nitrile production is ammoxidation as a gas‐phase reaction at high temperature. Further popular approaches are substitution or addition reactions with hydrogen cyanide or derivatives thereof. A major drawback, however, is the very high toxicity of cyanide. Recently, as a synthetic alternative, a novel enzymatic approach towards nitriles has been developed with aldoxime dehydratases, which are capable of converting an aldoxime in one step through dehydration into nitriles. Because the aldoxime substrates are easily accessible, this route is of high interest for synthetic purposes. However, whenever a novel method is developed for organic synthesis, it raises the question of substrate scope as one of the key criteria for application as a “synthetic platform technology”. Thus, the scope of this review is to give an overview of the current state of the substrate scope of this enzymatic method for synthesizing nitriles with aldoxime dehydratases. As a recently emerging enzyme class, a range of substrates has already been studied so far, comprising nonchiral and chiral aldoximes. This enzyme class of aldoxime dehydratases shows a broad substrate tolerance and accepts aliphatic and aromatic aldoximes, as well as arylaliphatic aldoximes. Furthermore, aldoximes with a stereogenic center are also recognized and high enantioselectivities are found for 2‐arylpropylaldoximes, in particular. It is further noteworthy that the enantiopreference depends on the E and Z isomers. Thus, opposite enantiomers are accessible from the same racemic aldehyde and the same enzyme.     

Rational Biocatalyst Design for a Cyanide-Free Synthesis of Long-Chain Fatty Nitriles from Their Aldoximes

Recently, the capability of the aldoxime dehydratase from Bacillus sp. (OxdB) for the transformation of fatty aldoximes into fatty nitriles with impressive substrate loadings is reported. However, the substrate scope of this biocatalyst turned out to be limited in terms of the chain length with decanal oxime being the substrate with the longest well tolerated n-alkyl chain. Besides the increased bulkiness of the long-chain aldoximes, their strongly decreased water solubility represents a further hurdle for an efficient biotransformation. Addressing this challenge of an expanded substrate spectrum comprising long-chain fatty aldoximes, this work investigates the substrate solubility and enzyme kinetics in combination with molecular modeling in order to find an enzyme mutant being suitable for C12- to C16-aldoximes. Both, fatty aldoxime solubility in water and the active site of the wild-type enzyme OxdB are identified as critical issues for an efficient biotransformation of these substrates. The activity issue is addressed by a rational design of a mutant using a homology modeling as well as a molecular modeling software suitable for enzymes. With the resulting double mutant OxdB-F289A/L293A, this report can achieve successful biotransformations with the C12- to C16-aldoximes at substrate concentrations of 250 × 10⁻³ to 1000 × 10⁻³ m . For example, an excellent conversion of >99% is obtained with tetradecanal oxime. Practical applications: Fatty nitriles with a prolonged chain length of C12 or more are of high interest in industry due to their use for the production of fatty amines on large technical scale. As an alternative route, fatty nitriles can be generated from their aldoximes by means of an aldoxime dehydratase (Oxd) as biocatalyst. The conversion of long-chain fatty aldoximes, however, remained a challenge up to now. This work describes the optimization of the aldoxime dehydratase OxdB from Bacillus sp. for the dehydration of nonsoluble bulky fatty aldoximes. The created variant can convert long chain fatty aldoximes toward the corresponding nitrile as demonstrated for C12- to C16-nitriles. In addition, high conversion (of up to >99%) is achieved when operating at high substrate concentrations of up to 1000 × 10⁻³ m , thus making this approach interesting for industrial applications.     

Optimization of nitrilase production from a new thermophilic isolate

A new thermostable nitrilase‐producing isolate identified as Streptomyces sp. MTCC 7546 has been studied extensively for the optimization of enzyme production operating in batch mode. The benzonitrile was observed as inducer of nitrilase production. The isolate showed maximum nitrilase production after 24 h of incubation at optimal conditions. The strain grows well on a variety of carbon sources and produces the nitrilase that catalyses the hydrolysis of nitriles to acids without formation of amides. The enzyme is mostly active against mono‐ and di‐aliphatic nitriles (10 mmol L^?1) at pH of 7.4 and at a temperature of 50 °C. Copyright © 2007 Society of Chemical Industry     

游离细胞腈水合酶催化丙烯腈水合反应的双稳态反应动力学

腈水合酶是能够催化丙烯腈水合生成丙烯酰胺的一种重要的工业酶。本研究建立了游离细胞腈水合酶催化丙烯腈水合反应的双稳态反应动力学模型,关联了底物浓度、产物浓度和温度等主要因素对反应速率(表观酶活)的影响。在实验研究的基础上,通过麦夸特及全局最优化算法求解了动力学模型。结果表明,游离细胞腈水合酶催化的双稳态反应动力学模型是比较典型的产物抑制型,当产物浓度逐渐增大时,高浓度的产物将抑制腈水合酶的活性。当底物浓度10g·L-1时,由于底物加入反应体系时产生的局部瞬时高浓度,腈水合酶催化的丙烯腈水合反应的表观反应速率不随底物浓度变化。当底物浓度≥10g·L-1时,底物产物浓度对反应速率具有显著影响。温度对酶活的影响也十分显著,相同底物产物浓度下,28℃时的酶催化水合反应速率是15℃时的3.3倍。     

一种腈水解酶的高通量筛选方法

水解腈的酶类(腈水解酶或腈水合酶/酰胺酶)在制药行业中生产有机羧酸及其衍生物方面有着广泛的应用,为了获得较多的酶源,建立高通量筛选方法是非常必要的.今利用改进的羟肟酸铁分光光度比色法建立了一种简单、快速、高通量的筛选方法,同时利用产酶菌株Rhodococcus sp. CCZU10-1静息细胞催化反应来进行方法的准确性验证.并且通过与液相色谱法检测结果进行对比,结果表明羟肟酸铁比色法对有机羧酸的检测具有较高的准确性.因此,建立的高通量筛选方法在产腈水解酶微生物筛选及应用方面有很大应用前景.     

Production of hydroxy fatty acids from unsaturated fatty acids byFlavobacterium sp. DS5 hydratase,a C-10 positional- andcis unsaturation-specific enzyme

A new microbial isolate,Flavobacterium sp. DS5, converted oleic and linoleic acids to their corresponding 10-keto-and 10-hydroxy fatty acids. The hydration enzyme seems to be specific to the C-10 position. Conversion products from α- and γ-linolenic acids were identified by gas chromatography/mass spectrometry, Fourier transform infrared, and nuclear magnetic resonance as 10-hydroxy-12(Z),15(Z)-octadecadienoic and 10-hydroxy-6(Z),12(Z)-octadecadienoic acids, respectively. Products from other 9(Z)-unsaturated fatty acids also were identified as their corresponding 10-hydroxy- and 10-keto-fatty acids.Trans unsaturated fatty acid was not converted. From these results, it is concluded that strain DS5 hydratase is indeed a C-10 positional-specific andcis-specific enzyme. DS5 hydratase prefers an 18-carbon monounsaturated fatty acid. Among the C₁₈ unsaturated fatty acids, an additional double bond at either side of the 9,10-position lowers the enzyme hydration activity. Because hydratases from other microbes also convert 9(Z)-unsaturated fatty acids to 10-hydroxy fatty acids, the C-10 positional specificity of microbial hydratases may be universal.     

Unexpected NADPH Hydratase Activity in the Nitrile Reductase QueF from Escherichia coli

The nitrile reductase QueF catalyzes NADPH-dependent reduction of the nitrile group of preQ₀ (7-cyano-7-deazaguanine) into the primary amine of preQ₁ (7-aminomethyl-7-deazaguanine), a biologically unique reaction important in bacterial nucleoside biosynthesis. Here we have discovered that the QueF from Escherichia coli—its D197A and E89L variants in particular (apparent k_cat≈10⁻² min⁻¹)—also catalyze the slow hydration of the C5=C6 double bond of the dihydronicotinamide moiety of NADPH. The enzymatically C6-hydrated NADPH is a 3.5:1 mixture of R and S forms and rearranges spontaneously through anomeric epimerization (β→α) and cyclization at the tetrahydronicotinamide C6 and the ribosyl O2. NADH and 1-methyl- or 1-benzyl-1,4-dihydronicotinamide are not substrates of the enzymatic hydration. Mutagenesis results support a QueF hydratase mechanism, in which Cys190—the essential catalytic nucleophile for nitrile reduction—acts as the general acid for protonation at the dihydronicotinamide C5 of NADPH. Thus, the NADPH hydration in the presence of QueF bears mechanistic resemblance to the C=C double bond hydration in natural hydratases.     

The thermal decomposition of polyurethanes and polyisocyanurates

The thermal decomposition of a number of TDI- and MDI-based biscarbamates (model compounds for polyurethane foams) between 200°C and 1000°C showed that the urethane linkage undergoes an O-acyl fission at about 300°C to generate the free isocyanate and alcohol. In the case of the flexible foam analogues, the newly generated TDI reacts further to generate volatile polyureas, termed ‘yellow smoke’. The MDI residues generated in the decomposition of a rigid foams react to yield non-volatile polycarbodiimides. Both the yellow smokes and the polycarbodiimides decompose above 600°C to give a mixture of nitriles (including HCN) as well as a number of olefinic and aromatic compounds. The use of ¹³C labeling indicated that HCN and all the other nitriles generated during the high temperature decompositions originate in the thermal fission of the aromatic ring, the nitrile carbon being the 2-, 4- or 6- carbon of MDI.     

Altered Glucosinolate Hydrolysis in Genetically Engineered Arabidopsis thaliana and its Influence on the Larval Development of Spodoptera littoralis

The antiherbivore potential of the glucosinolate–myrosinase defense system found in plants of the order Capparales is heavily influenced by the types of hydrolysis products (e.g. isothiocyanates, nitriles) formed from the parent glucosinolates upon plant damage. However, comparison of the effects of glucosinolate hydrolysis products on insect herbivores has been hampered by the lack of suitable experimental tools for rigorous bioassays, such as intact plants differing only in the types of hydrolysis products they produce, or artificial diets that can accurately simulate glucosinolate hydrolysis. The wide array of molecular resources for Arabidopsis thaliana has facilitated the identification of several genes that play a role in glucosinolate hydrolysis. One of these encodes the epithio-specifier protein (ESP) that promotes the formation of nitriles at the expense of isothiocyanates in certain ecotypes of A. thaliana. We overexpressed the ESP cDNA from the nitrile-producing ecotype Landsberg erecta in the isothiocyanate-producing ecotype Columbia-0 to generate transgenic lines of A. thaliana that differed from wild-type plants in the type of glucosinolate hydrolysis products formed upon tissue damage, whereas parent glucosinolate profile and myrosinase activity levels, as well as plant morphology and growth habit, remained unchanged. Bioassays with the model generalist herbivore Spodoptera littoralis (Lepidoptera: Noctuidae) demonstrated that larvae reared on the nitrile-producing lines on average gained weight faster in the first larval stages than larvae that fed on isothiocyanate-producing control plants. Furthermore, larvae with medial growth rates showed a tendency to pupate earlier on the ESP-overexpressing plant lines. Together with the results of previous studies, these findings suggest that isothiocyanates are more effective defenses against insect herbivores than nitriles, and raise questions about what conditions select for nitrile formation in plants.     

Optimization of Triazine Nitriles as Rhodesain Inhibitors: Structure–Activity Relationships,Bioisosteric Imidazopyridine Nitriles,and X‐ray Crystal Structure Analysis with Human Cathepsin L

The cysteine protease rhodesain of Trypanosoma brucei parasites causing African sleeping sickness has emerged as a target for the development of new drug candidates. Based on a triazine nitrile moiety as electrophilic headgroup, optimization studies on the substituents for the S1, S2, and S3 pockets of the enzyme were performed using structure‐based design and resulted in inhibitors with inhibition constants in the single‐digit nanomolar range. Comprehensive structure–activity relationships clarified the binding preferences of the individual pockets of the active site. The S1 pocket tolerates various substituents with a preference for flexible and basic side chains. Variation of the S2 substituent led to high‐affinity ligands with inhibition constants down to 2 nM for compounds bearing cyclohexyl substituents. Systematic investigations on the S3 pocket revealed its potential to achieve high activities with aromatic vectors that undergo stacking interactions with the planar peptide backbone forming part of the pocket. X‐ray crystal structure analysis with the structurally related enzyme human cathepsin L confirmed the binding mode of the triazine ligand series as proposed by molecular modeling. Sub‐micromolar inhibition of the proliferation of cultured parasites was achieved for ligands decorated with the best substituents identified through the optimization cycles. In cell‐based assays, the introduction of a basic side chain on the inhibitors resulted in a 35‐fold increase in antitrypanosomal activity. Finally, bioisosteric imidazopyridine nitriles were studied in order to prevent off‐target effects with unselective nucleophiles by decreasing the inherent electrophilicity of the triazine nitrile headgroup. Using this ligand, the stabilization by intramolecular hydrogen bonding of the thioimidate intermediate, formed upon attack of the catalytic cysteine residue, compensates for the lower reactivity of the headgroup. The imidazopyridine nitrile ligand showed excellent stability toward the thiol nucleophile glutathione in a quantitative in vitro assay and fourfold lower cytotoxicity than the parent triazine nitrile.     

Efficient Biocatalytic Synthesis of Highly Enantiopure α-Alkylated Arylglycines and Amides

A number of racemic α-alkylarylglycine amides including 1-amino-1-carbamoyl-1,2,3,4-tetrahydronaphthalene underwent efficient biocatalytic hydrolysis under very mild conditions to afford the corresponding (S)-α-alkylarylglycines and (R)-α-alkylarylglycine amides in excellent yields with enantiomeric excesses higher than 99.5%. Both the reaction rate and enantioselectivity of biocatalytic kinetic resolution were strongly dependent upon the nature of the substituent and the substitution pattern on the benzene ring of the substrate. In contrast, no effective biotransformation of the Strecker nitrile derived from acetophenone was observed under the catalysis of a nitrile hydratase/amidase-containing microbial Rhodococcus sp. AJ270 whole-cell catalyst. Coupled with the chemical hydrolysis of amide, this biotransformation process provided efficient syntheses of α-substituted arylglycines in both enantiomeric forms from readily available racemic amides.     

一株腈化物降解菌的分离、鉴定及降解特性

从长期被腈化物污染的土壤中分离到一株具有较宽腈化物降解利用谱的菌株YL-1。经过对YL-1形态特征及生理生化指标的分析,初步鉴定为红球菌。YL-1能降解丙烯腈生成丙烯酰胺。YL-1培养40h后可获得54U/mg的干细胞比活力。YL-1降解丙烯腈的最佳条件是温度30℃,pH=7 0,在该条件下对φ(丙烯腈)=0 2%的降解率可达99 4%。     

Selective hydrogenation of nitriles to imines over a multifunctional heterogeneous Pt catalyst

Imines and their derivatives are versatile synthetic intermediates for the industrial preparation of both bulk and fine chemicals and for pharmaceuticals, but preparing these compounds efficiently through direct hydrogenation of nitriles are hindered by overhydrogenation to secondary amines. Here we report a highly efficient multifunctional catalyst system for selective hydrogenation coupling of nitriles to secondary imines using a heterogeneous Pt catalyst that was deposited on a nickel‐based metal‐organic framework (MOF) containing DABCO. The catalyst showed excellent synergy in promoting the hydrogenation of a variety of nitriles, giving significantly improved activity and selectivity (up to >99% yield) even under atmospheric pressure of H₂. It is suggested that the Lewis base (DABCO) sites on the Ni‐MOF inhibit further hydrogenation of the imines. The influence of H₂ pressure, reactant concentration, stirring speed, and reaction temperature was investigated. The kinetics and mechanism of hydrogenation of benzonitrile (BN) by the Pt/Ni‐MOF catalyst has been studied. The reaction showed a first‐order dependence on both BN concentration and H₂ pressure. A kinetic model was proposed based on the mechanism of nitriles hydrogenation and compared with experimental observations. © 2014 American Institute of Chemical Engineers AIChE J, 60: 3565–3576, 2014     

一株产腈水合酶菌株酶活高效表达条件的研究

以抚顺腈纶厂废水中筛选的一株Nocardia sp为出发菌株,通过逐渐增加培养基中丙烯腈的体积浓度重复继代培养,得到1株酶活高效表达的Nocardia sp菌,酶活比出发菌株提高了15倍。确定了菌株培养的最优条件:葡萄糖(25 g/L),诱导剂脲(0.06 g/L)、Co2+(0.02 g/L)、丙烯酰胺(10%),反应条件pH(7)、温度(20℃),底物丙烯腈体积分数(<5%),产物丙烯酰胺的质量分数(<20%)。正交实验表明,反应温度和脲的加入量为反应过程中的显著因素。     

A Single Mutation Increases the Activity and Stability of Pectobacterium carotovorum Nitrile Reductase

Nitrile reductases are considered to be promising and environmentally benign nitrile‐reducing biocatalysts to replace traditional metal catalysts. Unfortunately, the catalytic efficiencies of the nitrile reductases reported so far are very low. To date, all attempts to increase the catalytic activity of nitrile reductases by protein engineering have failed. In this work, we successfully increased the specific activity of a nitrile reductase from Pectobacterium carotovorum from 354 to 526 U g_prot^?1 by engineering the substrate binding pocket; moreover, the thermostability was also improved (≈2‐fold), showing half‐lives of 140 and 32 h at 30 and 40 °C, respectively. In the bioreduction of 2‐amino‐5‐cyanopyrrolo[2,3‐d]pyrimidin‐4‐one (preQ₀) to 2‐amino‐5‐aminomethylpyrrolo[2,3‐d]pyrimidin‐4‐one (preQ₁), the variant was advantageous over the wild‐type enzyme with a higher reaction rate and complete conversion of the substrate within a shorter period. Homology modeling and docking analysis revealed some possible origins of the increased activity and stability. These results establish a solid basis for future engineering of nitrile reductases to increase the catalytic efficiency further, which is a prerequisite for applying these novel biocatalysts in synthetic chemistry.     

Characterization of membrane-bound lipase from a thermophilic Rhizopus oryzae isolated from palm oil mill effluent

The characteristics of the membrane-bound lipase from a thermophilic Rhizopus oryzae were studied. The pH and temperature optima for lipase activity were at 7.0 and 37°C, respectively. The enzyme was stable and acidic conditions, retaining more than 80% of its initial activity at pH 4.0 after 30 min incubation. It was stable up to 50°C with 70% of initial activity retained after 3 h incubation. The enzyme is 1,3 specific and exhibits substrate preference. Monoacid triglyceride substrates were hydrolyzed better than methyl esters, polyoxysorbitan and sorbitan substrates.     

Kinetic study of the enantioselective hydrolysis of a meso‐diester using pig liver esterase

A kinetic study of the hydrolysis of the diester dimethyl cis‐cyclohex‐4‐ene‐1,2‐dicarboxylate, to the (1S,2R)‐monoester, catalysed by the enzyme Pig Liver Esterase (PLE) was performed. The effects of the most relevant parameters that influence the enzymatic conversion were studied, such as pH, temperature and concentration of substrate and reaction products. It was concluded that the pH at which the enzyme exhibits a maximum activity is pH 7. At 25 °C PLE presents a better long‐term stability and enantioselectivity than at higher temperatures, although the reaction rate is slower. The kinetic results obtained are well described by the Michaelis–Menten equation, although a slight deviation to this model was observed for low substrate concentrations. Methanol, a co‐product of the enzymatic hydrolysis, was found to act as a non‐competitive inhibitor of the reaction. The Michaelis–Menten parameters were determined and a comprehensive kinetic model, which already accounts for methanol inhibition, is presented. © 2000 Society of Chemical Industry