Enhancement Of Catalytic Efficiency Research Articles

Improving Glyphosate Oxidation Activity of Glycine Oxidase from Bacillus cereus by Directed Evolution

Glyphosate, a broad spectrum herbicide widely used in agriculture all over the world, inhibits 5-enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway, and glycine oxidase (GO) has been reported to be able to catalyze the oxidative deamination of various amines and cleave the C-N bond in glyphosate. Here, in an effort to improve the catalytic activity of the glycine oxidase that was cloned from a glyphosate-degrading marine strain of Bacillus cereus (BceGO), we used a bacteriophage T7 lysis-based method for high-throughput screening of oxidase activity and engineered the gene encoding BceGO by directed evolution. Six mutants exhibiting enhanced activity toward glyphosate were screened from two rounds of error-prone PCR combined with site directed mutagenesis, and the beneficial mutations of the six evolved variants were recombined by DNA shuffling. Four recombinants were generated and, when compared with the wild-type BceGO, the most active mutant B3S1 showed the highest activity, exhibiting a 160-fold increase in substrate affinity, a 326-fold enhancement in catalytic efficiency against glyphosate, with little difference between their pH and temperature stabilities. The role of these mutations was explored through structure modeling and molecular docking, revealing that the Arg51 mutation is near the active site and could be an important residue contributing to the stabilization of glyphosate binding, while the role of the remaining mutations is unclear. These results provide insight into the application of directed evolution in optimizing glycine oxidase function and have laid a foundation for the development of glyphosate-tolerant crops.

Activity Enhancement of Candida antarctica Lipase B by Flexibility Modulation in Helix Region Surrounding the Active Site

The activity of Candida antarctica lipase B was improved by mutation of the area surrounding the active site. We changed the edges of four helices surrounding the active site to flexible amino acids. Two mutants, V139E and I255E, obtained as a result of Pichia pastoris expression, showed enhanced specific activity of 9.9 and 8.1 U/mg while that of wild type was 2.3 U/mg for p-nitrophenyl caprylate hydrolysis. It was nearly 5.4-fold and 3.5-fold, respectively. The stability of both mutants on organic solvent was slightly decreased but almost similar with that of wild type. In the kinetic assay, k(cat) values were shown as dominant factor for the enhancement of catalytic efficiency, k(cat)/K(m), since it was 4.1-fold and 3.8-fold, respectively.

ChemInform Abstract: Catalytic Asymmetric Intermolecular Stetter Reaction of Enals with Nitroalkenes: Enhancement of Catalytic Efficiency Through Bifunctional Additives.

AbstractThe title reaction is performed with aromatic and aliphatic enals in the presence of chiral fluorinated triazolium salt FTB as precatalyst and catechols as additives, which significantly enhance the reactivity and chemical yields.

Catalytic Asymmetric Intermolecular Stetter Reaction of Enals with Nitroalkenes: Enhancement of Catalytic Efficiency through Bifunctional Additives

An asymmetric intermolecular Stetter reaction of enals with nitroalkenes catalyzed by chiral N-heterocyclic carbenes has been developed. The reaction rate and efficiency are profoundly impacted by the presence of catechol. The reaction proceeds with high selectivities and affords good yields of the Stetter product. Internal redox products were not observed despite of the protic conditions. The impact of catechol has been found to be general, facilitating far lower catalyst loadings than were previously achievable.

Lactose nano-probe optimized using response surface methodology

A lactose nano-probe has been developed by immobilization of PsBGAL onto gold nanoparticles (AuNps). It is helpful for severe lactose intolerants for quality check of lactose hydrolyzed milk and estimation of hidden lactose present in variety of food products. Optimization of PsBGAL immobilization onto AuNps using spacer arm (cysteamine–glutaraldehyde) was carried out by response surface methodology (Box–Behnken design). The process has led to immobilization of enzyme onto AuNps with an efficiency of 140.81%. AuNp–PsBGAL was characterized using transmission electron microscopy, scanning electron microscopy and Fourier transform infrared spectroscopy. Immobilized enzyme showed broad temperature and pH optima and a significant enhancement in catalytic efficiency (Vmax/Km) with respect to soluble PsBGAL. AuNp–PsBGAL was stable under dried conditions than wet conditions for 6 months with loss of 10.2% and 87.53%, respectively. It has reusability of over five batchwise uses, with almost no loss in activity. Hill's coefficient was found to be 1.71 corresponding to lactose concentration ranging from 0.1% to 2.0%.

Chemically Modified, Immobilized Trypsin Reactor with Improved Digestion Efficiency

Tryptic digestion followed by identification using mass spectrometry is an important step in many proteomic studies. Here, we describe the preparation of immobilized, acetylated trypsin for enhanced digestion efficacy in integrated protein analysis platforms. Complete digestion of cytochrome c was obtained with two types of modified-trypsin beads with a contact time of only 4 s, while corresponding unmodified-trypsin beads gave only incomplete digestion. The digestion rate of myoglobin, a protein known to be rather resistant to proteolysis, was not altered by acetylating trypsin and required a buffer containing 35% acetonitrile to obtain complete digestion. The use of acetylated-trypsin beads led to fewer interfering tryptic autolysis products, indicating an increased stability of this modified enzyme. Importantly, the modification did not affect trypsin's substrate specificity, as the peptide map of myoglobin was not altered upon acetylation of immobilized trypsin. Kinetic digestion experiments in solution with low-molecular-weight substrates and cytochrome c confirmed the increased catalytic efficiency (lower K(M) and higher k(cat)) and increased resistance to autolysis of trypsin upon acetylation. Enhancement of catalytic efficiency was correlated with the number of acetylations per molecule. The favorable properties of the new chemically modified trypsin reactor should make it a valuable tool in automated protein analysis systems.

Role of Plasminogen-Fibrin Interaction in Plasminogen Activation by t-PA.

Plasminogen (Pg) is the zymogen of the fibrinolytic enzyme plasmin (Pn). Fibrin (Fn) promotes the activation of Pg by tissue-type plasminogen activator (t-PA) by serving as a template that brings t-PA and Pg into close proximity. In addition, proteolysis of Fn by Pn generates carboxyl-terminal lysine residues that provide nascent high affinity binding sites for Glu-Pg, thereby promoting Pg activation. Pg activation is also enhanced when Glu-Pg is converted to Lys-Pg, a derivative with higher Fn affinity. Therefore, kinetic and functional data suggest that Pg binding to Fn is a key aspect of efficient Pg activation. To explore this concept, we performed binding and kinetic analyses with Mini-Pg, an elastase-derived fragment of Glu-Pg with reduced Fn affinity. Binding of Glu-Pg, Lys-Pg and Mini-Pg to immobilized fibrinogen (Fg) and fibrin monomer (Fm) was monitored by surface plasmon resonance. The affinity of Glu-Pg for Fm is 4-fold higher than that for Fg with Kd values of 3.1 and 12.5 μM, respectively, whereas Lys-Pg binds with high affinity to both Fg and Fm (Kd values of 0.25 and 0.21 μM, respectively). In contrast, Mini-Pg demonstrates weak binding to Fg and Fm with Kd values of 10.5 and 24.5 μM, respectively. To complement the binding experiments, kinetic studies of Pg activation by t-PA were performed in the absence or presence of native Fn clots by monitoring Pn formation using a Pn-directed chromogenic substrate. As expected, the catalytic efficiency of Lys-Pg or Mini-Pg activation by t-PA in the absence of cofactor was higher than that of Glu-Pg (kcat/KM values of 1.3, 0.325, and 0.026 μM−1 min−1, respectively). The catalytic efficiency of Glu-Pg activation by t-PA is 500-fold higher in the presence of Fn than it is in its absence. The stimulatory effect of fibrin was maintained with Lys and Mini-Pg with over 100-fold enhancement in catalytic efficiency of activation. The fibrin-dependent increase in catalytic efficiency was expressed predominantly through a decrease in KM, with values of >20 and 0.2 μM for Glu-Pg activation in the absence and presence of fibrin, respectively. Lys and Mini-Pg also expressed similar enhancements in catalytic efficiency through a decrease in KM. Thus, despite a 100-fold range in their affinities for Fn, the activation of Mini-Pg, Glu-Pg and Lys-Pg by t-PA are all enhanced by at least 2 orders of magnitude in the presence of Fn. These results demonstrate that substrate binding to Fn is not essential for Fn-mediated stimulation of Pg activation by t-PA. To investigate the importance of the Fn-plasminogen activator interaction, activation studies were carried out using urokinase-type plasminogen activator (u-PA), an activator without Fn affinity. In contrast to the results with t-PA, Fn did not enhance u-PA-mediated activation of Glu, Lys or Mini-Pg. The lack of fibrin stimulation of Pg activation by u-PA suggests that the Pg-Fn interaction is not essential to Pg activation. Therefore, the cofactor role of Fn is expressed predominantly through interaction and stimulation of t-PA. These findings support the hypothesis that Fn binding to t-PA may expose cryptic binding sites in the activator that stabilize the formation of the enzyme-substrate complex.

Multivalency and the mode of action of bacterial sialidases.

Although complex modular proteins are encountered frequently in a variety of biological systems, their occurrence in biocatalysis has not been widely appreciated. Here, we describe that bacterial sialidases, which have both a catalytic and carbohydrate-binding domain, can hydrolyze polyvalent substrates with much greater catalytic efficiency than their monovalent counterparts. The enhancement of catalytic efficiency was due to a much smaller Michaelis constant and rationalized by a model in which the catalytic and lectin domains interact simultaneously with the polyvalent substrate, leading to an enhancement of affinity. Inhibition studies have shown that galactosides released by the action of the sialidase can act as the ligand for the lectin domain. This knowledge has been exploited in the design of a potent polyvalent inhibitor of the sialidase of Vibrio cholerae, which displayed exquisite selectivities for sialidases that have a lectin domain.

Conversion of a glutamate dehydrogenase into methionine/norleucine dehydrogenase by site‐directed mutagenesis

In earlier attempts to shift the substrate specificity of glutamate dehydrogenase (GDH) in favour of monocarboxylic amino-acid substrates, the active-site residues K89 and S380 were replaced by leucine and valine, respectively, which occupy corresponding positions in leucine dehydrogenase. In the GDH framework, however, the mutation S380V caused a steric clash. To avoid this, S380 has been replaced with alanine instead. The single mutant S380A and the combined double mutant K89L/S380A were satisfactorily overexpressed in soluble form and folded correctly as hexameric enzymes. Both were purified successfully by Remazol Red dye chromatography as routinely used for wild-type GDH. The S380A mutant shows much lower activity than wild-type GDH with glutamate. Activities towards monocarboxylic substrates were only marginally altered, and the pH profile of substrate specificity was not markedly altered. In the double mutant K89L/S380A, activity towards glutamate was undetectable. Activity towards L-methionine, L-norleucine and L-norvaline, however, was measurable at pH 7.0, 8.0 and 9.0, as for wild-type GDH. Ala163 is one of the residues that lines the binding pocket for the side chain of the amino-acid substrate. To explore its importance, the three mutants A163G, K89L/A163G and K89L/S380A/A163G were constructed. All three were abundantly overexpressed and showed chromatographic behaviour identical with that of wild-type GDH. With A163G, glutamate activity was lower at pH 7.0 and 8.0, but by contrast higher at pH 9.0 than with wild-type GDH. Activities towards five aliphatic amino acids were remarkably higher than those for the wild-type enzyme at pH 8.0 and 9.0. In addition, the mutant A163G used L-aspartate and L-leucine as substrates, neither of which gave any detectable activity with wild-type GDH. Compared with wild-type GDH, the A163 mutant showed lower catalytic efficiencies and higher K(m ) values for glutamate/2-oxoglutarate at pH 7.0, but a similar k(cat)/K(m) value and lower K(m) at pH 8.0, and a nearly 22-fold lower S(0.5) (substrate concentration giving half-saturation under conditions where Michaelis-Menten kinetics does not apply) at pH 9.0. Coupling the A163G mutation with the K89L mutation markedly enhanced activity (100-1000-fold) over that of the single mutant K89L towards monocarboxylic amino acids, especially L-norleucine and L-methionine. The triple mutant K89L/S380A/A163G retained a level of activity towards monocarboxylic amino acids similar to that of the double mutant K89L/A163G, but could no longer use glutamate as substrate. In terms of natural amino-acid substrates, the triple mutant represents effective conversion of a glutamate dehydrogenase into a methionine dehydrogenase. Kinetic parameters for the reductive amination reaction are also reported. At pH 7 the triple mutant and K89L/A163G show 5 to 10-fold increased catalytic efficiency, compared with K89L, towards the novel substrates. In the oxidative deamination reaction, it is not possible to estimate k(cat) and K(m) separately, but for reductive amination the additional mutations have no significant effect on k(cat) at pH 7, and the increase in catalytic efficiency is entirely attributable to the measured decrease in K(m). At pH 8 the enhancement of catalytic efficiency with the novel substrates was much more striking (e.g. for norleucine approximately 2000-fold compared with wild-type or the K89L mutant), but it was not established whether this is also exclusively due to more favourable Michaelis constants.

Characterization of the glutathione binding site of aldose reductase

Despite extensive investigations, the physiological role of the polyol pathway enzyme–aldose reductase (AR) remains obscure. While the enzyme reduces glucose in vivo and in vitro, kinetic and structural studies indicate inefficient carbohydrate binding to the active site of the enzyme. The active site is lined by hydrophobic residues and appears more compatible with the binding of medium- to long-chain aliphatic aldehydes or hydrophobic aromatic aldehydes. In addition, our recent studies show that glutathione (GS) conjugates are also reduced efficiently by the enzyme. For instance, the GS conjugate of acrolein is reduced with a catalytic efficiency 1000-fold higher than the parent aldehyde, indicating specific recognition of glutathione by the active site residues of AR. An increase in the catalytic efficiency upon glutathiolation was also observed with trans-2-nonenal, trans-2-hexenal and trans, trans-2,4-decadienal, establishing that enhancement of catalytic efficiency was specifically due to the glutathione backbone and not specific to the aldehyde. Structure–activity relationships with substitution or deletion of amino acids of GSH indicated specific interactions of the active site with γ-Glu1 and Cys of GSH. Molecular modeling revealed that the glutathione–propanal conjugate could bind in two distinct orientations. In orientation 1, γ-Glu1 of the conjugate interacts with Trp20, Lys21 and Val47, and Gly3 interacts with Ser302 and Leu301, whereas in orientation 2, the molecule is inverted with γ-Glu1 interacting with Ser302, and Leu301. Taken together, these data suggest that glutathiolation of aldehydes enhances their compatibility with the AR active site, which may be of physiological significance in detoxification of endogenous and xenobiotic aldehydes.

Change of nucleotide specificity and enhancement of catalytic efficiency in single point mutants of Vibrio harveyi aldehyde dehydrogenase.

The fatty aldehyde dehydrogenase from the luminescent bacterium, Vibrio harveyi (Vh-ALDH), is unique with respect to its high specificity for NADP(+) over NAD(+). By mutation of a single threonine residue (Thr175) immediately downstream of the beta(B) strand in the Rossmann fold, the nucleotide specificity of Vh-ALDH has been changed from NADP(+) to NAD(+). Replacement of Thr175 by a negatively charged residue (Asp or Glu) resulted in an increase in k(cat)/K(m) for NAD(+) relative to that for NADP(+) of up to 5000-fold due to a decrease for NAD(+) and an increase for NADP(+) in their respective Michaelis constants (K(a)). Differential protection by NAD(+) and NADP(+) against thermal inactivation and comparison of the dissociation constants of NMN, 2'-AMP, 2'5'-ADP, and 5'-AMP for these mutants and the wild-type enzyme clearly support the change in nucleotide specificity. Moreover, replacement of Thr175 with polar residues (N, S, or Q) demonstrated that a more efficient NAD(+)-dependent enzyme T175Q could be created without loss of NADP(+)-dependent activity. Analysis of the three-dimensional structure of Vh-ALDH with bound NADP(+) showed that the hydroxyl group of Thr175 forms a hydrogen bond to the 2'-phosphate of NADP(+). Replacement with glutamic acid or glutamine strengthened interactions with NAD(+) and indicated why threonine would be the preferred polar residue at the nucleotide recognition site in NADP(+)-specific aldehyde dehydrogenases. These results have shown that the size and the structure of the residue at the nucleotide recognition site play the key roles in differentiating between NAD(+) and NADP(+) interactions while the presence of a negative charge is responsible for the decrease in interactions with NADP(+) in Vh-ALDH.

Mediated Electrocatalysis with Polyanthraquinone-Functionalized Monolayer-Protected Clusters

This paper describes the reduction of 1,1-dinitrocyclohexane by electrogenerated anthraquinone radical anions that are ligated to monolayer-protected gold clusters. Each cluster bears multiple (ca. 18) anthraquinone units. The clusters are prepared by place-exchange of a fraction of the cluster's original octanethiolate ligands with 1-(1,3-dithiapropyl)anthracene-9,10-dione. The electrocatalytic reduction currents are compared to those observed for anthraquinone monomer at an equivalent concentration. The electrocatalytic efficiency is larger for cluster-bound anthraquinone. The results are analyzed by digital simulation and by a discrete mathematical approach so as to determine the rate constant for electron transfer between the substrate and anthraquinone radical anion (cluster-bound and free). The rate constant is slightly larger for the free anthraquinone monomer. The enhancement in catalytic efficiency for cluster-bound anthraquinone arises from the smaller diffusion coefficient of cluster-bound anthraquinone, relative to that of the monomer, resulting in spatial compression of the reaction zone next to the electrode.

Apolipoprotein B mRNA editing: the sequence to the event

Editing of the mRNA coding for apolipoprotein B involves a cytidine to uridine transition at nucleotide 6666 and enables translation of two protein variants. The development of in vitro editing systems has led to the identification of three sequence requirements in this process. The mechanism for C→U editing requires specific sequences for editing site recognition, positioning of the catalytic activity and enhancement of catalytic efficiency. The dependence of in vitro editing on extract proteins has focused future research in this field on the identification of factors involved in apoB mRNA editing and the role of these factors in the assembly of ribonucleoprotein editosomes.

Essential roles of alkylammonium and alkylguanidinium ions in trypsin-catalyzed hydrolysis of acetylglycine esters: enhancement of catalytic efficiency analyzed by the use of "inverse substrates".

An "inverse substrate" for trypsin, p-amidinophenyl acetylglycinate, has been synthesized and applied to mechanistic studies of tryptic hydrolysis. The compound reacts to form the intermediate acetylglycyl trypsin in a very specific manner, analogous to the reactions of p-amidinophenyl alkanoates with trypsin to give alkanoyl trypsins (Tanizawa, K., Kasaba, Y., & Kanaoka, Y. (1977) J. Am. Chem Soc. 99, 4485). The effects of alkylammonium and alkylguanidinium ions on tryptic hydrolysis of this "inverse substrate" have been investigated, and all alkylamines and alkylguanidines tested behaved as activators. n-Propylamine, n-propylguanidine, and n-butylguanidine, which have been reported to inhibit tryptic hydrolysis of ethyl or p-nitrophenyl acetylglycinate, were activators with p-amidinophenyl acetylglycinate. The activating properties of these amines and guanidines were demonstrated for the first time by making use of the specific characteristics of this inverse substrate. Tryptic mechanisms proposed in the literature are discussed in the light of these observations.

Hydrolysis and transpeptidation of peptide substrates by acetyl-pepsin.

Treatment of swine pepsin with acetylimidazole to acetylate approximately five of its 16 tyrosyl residues causes a significant enhancement of catalytic efficiency (kcat/Km) toward substrates such as dansyl-glycyl-glycyl-L-phenylalanyl-L-phenylalanine 3-(4-pyridyl)propyl ester and benzyloxy-carbonyl-(glycyl)n-p-nitroLphenylalnyl-Lphenylalanyl-L-tyrosine (where n = 0, 1,2). Stopped-flow kinetic studies, under conditions of enzyme excess, with the dansyl peptide have shown that, as with untreated pepsin, the rate-limiting step in the over-all catalytic process is associated with the decomposition of the first detectable enzyme-substrate complex, whose dissociation constant is approximately equal to the Km found in steady-state kinetic experiments. With substrates of the type benzoyl-(glycyl)n-nitro-L-phenylalanyl-L-tyrosine, an increase in the chain length of the peptide leads to an increase in the value of kcat/Km, supporting the view that secondary enzyme-substrate interactions may produce at the extended active site conformational changes that are reflected in higher catalytic efficiency. This effect is more marked with acetyl-pepsin than with untreated pepsin, and suggests that the conformational mobility of the active site is increased by partial acetylation. Acetyl-pepsin is less effective than untreated pepsin in catalyzing transpeptidation reactions in which acetyl-L-phenylalanyl-L-tyrosine and benzyloxycarbonyl-(glycyl)n-p-nitro-L-phenylalanine are the reactants; this finding is consistent with the more rapid hydrolysis of the product of transpeptidation.