Activity Of Mutant Enzyme Research Articles

Biocatalytic Synthesis of α-Amino Esters via Nitrene C-H Insertion.

α-Amino esters are precursors to noncanonical amino acids used in developing small-molecule therapeutics, biologics, and tools in chemical biology. α-C-H amination of abundant and inexpensive carboxylic acid esters through nitrene transfer presents a direct approach to α-amino esters. Methods for nitrene-mediated amination of the protic α-C-H bonds in carboxylic acid esters, however, are underdeveloped. This gap arises because hydrogen atom abstraction (HAA) of protic C-H bonds by electrophilic metal-nitrenoids is slow: metal-nitrenoids preferentially react with polarity-matched, hydridic C-H bonds, even when weaker protic C-H bonds are present. This study describes the discovery and evolution of highly stable protoglobin nitrene transferases that catalyze the enantioselective intermolecular amination of the α-C-H bonds in carboxylic acid esters. We developed a high-throughput assay to evaluate the activity and enantioselectivity of mutant enzymes together with their sequences using the Every Variant Sequencing (evSeq) method. The assay enabled the identification of enantiodivergent enzymes that function at ambient conditions in Escherichia coli whole cells and whose activities can be enhanced by directed evolution for the amination of a range of substrates.

Enhancing the Catalytic Activity of Geranylgeranyl Diphosphate Synthase through Ancestral Sequence Reconstruction and Semirational Design.

Geranylgeranyl diphosphate synthase (GGPPS) is the crucial bottleneck in carotenoid biosynthesis. However, low activity limits the broad application of GGPPS. In this study, OsGGPPS1 in rice was engineered based on ancestral sequence reconstruction (ASR) and semirational design to improve the catalytic performances of existing GGPPS. The better mutant of A22R/A26P with improved enzyme activity was generated based on ASR. Additionally, the improved enzyme activity of mutants as V162A/M218S/F227Y was designed using a semirational design. The combinatorial assembly of the d-OsGGPPS1 mutant (A22R/A26P/V162A/M218S/F227Y) exhibited higher conversion of IPP and each cosubstrate of DMAPP for 9.8-fold in GPP production, GPP for 6.4-fold in FPP production, and FPP for 1.4-fold in GGPP production relative to wild-type OsGGPPS1 at 25 °C, which showed higher conversion than wild-type OsGGPPS1 at temperatures as high as 50 °C. The successful design of OsGGPPS1 was representative of protein engineering, which will shed new light on GGPPS engineering and active plant pigment resource utilization.

Insights into the Mechanism of Catalytic Activity of Plasmodium Parasite Malate-Quinone Oxidoreductase.

Plasmodium malate-quinone oxidoreductase (MQO) is a membrane flavoprotein catalyzing the oxidation of malate to oxaloacetate and the reduction of quinone to quinol. Recently, using a yeast expression system, we demonstrated that MQO, expressed in place of mitochondrial malate dehydrogenase (MDH), contributes to the TCA cycle and the electron transport chain in mitochondria, making MQO attractive as a promising drug target in Plasmodium malaria parasites, which lack mitochondrial MDH. However, there is little information on the structure of MQO and its catalytic mechanism, information that will be required to develop novel drugs. Here, we investigated the catalytic site of P. falciparum MQO (PfMQO) using our yeast expression system. We generated a model structure for PfMQO with the AI tool AlphaFold and used protein footprinting by acetylation with acetic anhydride to analyze the surface topology of the model, confirming the computational prediction to be reasonably accurate. Moreover, a putative catalytic site, which includes a possible flavin-binding site, was identified by this combination of protein footprinting and structural prediction model. This active site was analyzed by site-directed mutagenesis. By measuring enzyme activity and protein expression levels in the PfMQO mutants, we showed that several residues at the active site are essential for enzyme function. In addition, a single substitution mutation near the catalytic site resulted in enhanced sensitivity to ferulenol, an inhibitor of PfMQO that competes with malate for binding to the enzyme. This strongly supports the notion that the substrate binds to the proposed catalytic site. Then, the location of the catalytic site was demonstrated by structural comparison with a homologous enzyme. Finally, we used our results to propose a mechanism for the catalytic activity of MQO by reference to the mechanism of action of structurally or functionally homologous enzymes.

Elucidating the Role of a Calcium-Binding Loop in an x-Prolyl Aminodipeptidase from Lb. helveticus.

Prolyl aminodipeptidase (PepX) is an α/β hydrolase that cleaves at penultimate N-terminal prolyl peptide bonds. The crystal structure of PepX from Lactobacillus helveticus exhibits a calcium-binding loop within the catalytic domain. The calcium-binding sequence of xDxDxDGxxD within this loop is highly conserved in PepX proteins among lactic acid bacteria, but its purpose remains unknown. Enzyme activity is not significantly affected in the presence of the metal chelator ethylenediaminetetraacetic acid (EDTA), nor in the presence of excess calcium ions. To eliminate calcium binding, D196A and D194A/D196A mutations were constructed within the conserved calcium-binding sequence motif. Enzyme activity and stability of the D196A mutant were comparable to the wild-type enzyme by colorimetric kinetic assays and protein thermal shift assays. However, the D194A/D196A mutant was inactive though it retained native-like structure and thermal stability, contradicting the EDTA and calcium titration results. This suggests calcium binding to PepX may be essential for activity.

Simultaneously Enhanced Thermostability and Catalytic Activity of Xylanase from Streptomyces rameus L2001 by Rigidifying Flexible Regions in Loop Regions of the N-Terminus.

The GH11 xylanase XynA from Streptomyces rameus L2001 has favorable hydrolytic properties. However, its poor thermal stability hinders its widespread application in industry. In this study, mutants Mut1 and Mut2 were constructed by rationally combining the mutations 11YHDGYF16, 23AP24/23SP24, and 32GP33. The residual enzyme activity of these combinational mutants was more than 85% when incubated at 80 and 90 °C for 12 h, and thus are the most thermotolerant xylanases known to date. The reduced flexibility of the N-terminus, increased overall rigidity, as well as the surface net charge of Mut1 and Mut2 may be partially responsible for the improved thermal stability. In addition, the specific activity and catalytic efficiency of Mut1 and Mut2 were improved compared with those of wild-type XynA. The broader catalytic cleft and enhanced flexibility of the "thumb" of Mut1 and Mut2 may be partially responsible for the improved specific activity and catalytic efficiency.

Improved thermostability of type I pullulanase from Bacillus thermoliquefaciens by error-prone PCR

Pullulanase (PulB) is a starch-debranching enzyme. In order to improve its catalytic performance, random mutagenesis was performed on the pullulanase gene derived from Bacillus thermoliquefaciens. Two rounds of error-prone PCR were carried out. Mutant T252S was screened in the first round of error-prone library, which had the highest catalytic activity. During the second round of mutations, mutant enzyme G250P/T252S/G253T/N255K was screened, which had further improved catalytic activity and the best thermostability. Compared with the parent enzyme, the specific activity of mutant enzyme G250P/T252S/G253T/N255K increased by 1.9 times, Km decreased by 22.7 %, kcat increased by 28.7 %, and kcat/Km increased by 68.4 %. The thermostability of the mutant enzyme improved significantly, showing that the half-life at 60 °C was extended to 7.5 h, which was 87.5 % higher than that of the parent enzyme. The mutation sites in these two rounds were concentrated in the 250–255 regions, indicating that this region was an important region affecting the catalytic activity and Thermostability. The reasons for the change of enzymtic properties was also preliminarily analyzed through three-dimensional simulation.

Site-directed mutagenesis improves the practical application of L-glutamic acid decarboxylase in Escherichia coli.

γ-Aminobutyric acid (GABA) is a kind of non-proteinogenic amino acid which is highly soluble in water and widely used in the food and pharmaceutical industries. Enzymatic conversion is an efficient method to produce GABA, whereby glutamic acid decarboxylase (GAD) is the key enzyme that catalyzes the process. The activity of wild-type GAD is usually limited by temperature, pH or biotin concentration, and hence directional modification is applied to improve its catalytic properties and practical application. GABA was produced using whole cell transformation of the recombinant strains Escherichia coli BL21(DE3)-Gad B, E. coli BL21(DE3)-Gad B-T62S and E. coli BL21(DE3)-Gad B-Q309A. The corresponding GABA concentrations in the fermentation broth were 219.09, 238.42, and 276.66g/L, and the transformation rates were 78.02%, 85.04%, and 98.58%, respectively. The results showed that Gad B-T62S and Gad B-Q309A are two effective mutation sites. These findings may contribute to ideas for constructing potent recombinant strains for GABA production. Practical Application : Enzymatic properties of the GAD from Escherichia coli and GAD site-specific mutants were examined by analyzing their conserved sequences, substrate contacts, contact between GAD amino acid residues and mutation energy (ΔΔG) of the GAD mutants. The enzyme activity and stability of Gad B-T62S and Gad B-Q309A mutants were improved compared to Gad B. The kinetic parameters Km and Vmax of Gad B, Gad B-T62S, and Gad B-Q309A mutants were 11.3 ± 2.1mM and 32.1 ± 2.4U/mg, 7.3 ± 2.5mM and 76.1 ± 3.1U/mg, and 7.2 ± 3.8mM and 87.3±1.1U/mg, respectively. GABA was produced using whole cell transformation of the recombinant strains E. coli BL21(DE3)-Gad B, E. coli BL21(DE3)-Gad B-T62S, and E. coli BL21(DE3)-Gad B-Q309A. The corresponding GABA concentrations in the fermentation broth were 219.09, 238.42, and 276.66g/L, and the transformation rates were 78.02%, 85.04%, and 98.58%, respectively.

Structural basis for heparan sulfate co-polymerase action by the EXT1-2 complex.

Heparan sulfate (HS) proteoglycans are extended (-GlcAβ1,4GlcNAcα1,4-)n co-polymers containing decorations of sulfation and epimerization that are linked to cell surface and extracellular matrix proteins. In mammals, HS repeat units are extended by an obligate heterocomplex of two exostosin family members, EXT1 and EXT2, where each protein monomer contains distinct GT47 (GT-B fold) and GT64 (GT-A fold) glycosyltransferase domains. In this study, we generated human EXT1-EXT2 (EXT1-2) as a functional heterocomplex and determined its structure in the presence of bound donor and acceptor substrates. Structural data and enzyme activity of catalytic site mutants demonstrate that only two of the four glycosyltransferase domains are major contributors to co-polymer syntheses: the EXT1 GT-B fold β1,4GlcA transferase domain and the EXT2 GT-A fold α1,4GlcNAc transferase domain. The two catalytic sites are over 90 Å apart, indicating that HS is synthesized by a dissociative process that involves a single catalytic site on each monomer.

Biochemical and genetic characterization of Botrytis cinerea laboratory mutants resistant to propamidine.

Botrytis cinerea, the causal agent of gray mold, is one of the top 10 fungal pathogens in the world. Propamidine, an aromatic diamidine compound, exhibited both protective and therapeutic effects against B. cinerea. However, the resistance risk and mechanism of B. cinerea to propamidine are unclear. Twelve high and stable resistant mutants were obtained from B. cinerea B05.10 by fungicide induction. Compared with the parental strain, the biological fitness of the mutants, including growth rate, spore germination, pathogenicity, and oxalic acid decreased significantly. There was no cross-resistance among propamidine and other commonly used fungicides, while the efficacy of propamidine against the resistance mutants declined. In addition, the cell membrane permeability, substance metabolism, and defense enzyme activities of the resistant mutants were significantly increased compared with the wild strain. Whole-genome sequencing of all resistant mutants found that there were 32 SNPs and nine InDels. Importantly, nine common single-point mutant genes in the exon region were found in all 12 resistant mutants, and these genes were related to multiple pathways in vivo, indicating that many factors contributed to the formation of propamidine resistance. These data suggested the resistance risk of B. cinerea to propamidine was low to moderate and the mechanism of propamidine was different from that of the existing fungicides. These results will increase understanding of the resistance mechanism of propamidine and provide a critical basis for the rational design of pesticide molecules based on targets. © 2022 Society of Chemical Industry.

A thermostable and CBM2-linked GH10 xylanase from Thermobifida fusca for paper bleaching.

Xylanases have the potential to be used as bio-deinking and bio-bleaching materials and their application will decrease the consumption of the chlorine-based chemicals currently used for this purpose. However, xylanases with specific properties could act effectively, such as having significant thermostability and alkali resistance, etc. In this study, we found that TfXyl10A, a xylanase from Thermobifida fusca, was greatly induced to transcript by microcrystalline cellulose (MCC) substrate. Biochemical characterization showed that TfXyl10A is optimally effective at temperature of 80 °C and pH of 9.0. After removing the carbohydrate-binding module (CBM) and linker regions, the optimum temperature of TfXyl10A-CD was reduced by 10°C (to 70°C), at which the enzyme’s temperature tolerance was also weakened. While truncating only the CBM domain (TfXyl10AdC) had no significant effect on its thermostability. Importantly, polysaccharide-binding experiment showed that the auxiliary domain CBM2 could specifically bind to cellulose substrates, which endowed xylanase TfXyl10A with the ability to degrade xylan surrounding cellulose. These results indicated that TfXyl10A might be an excellent candidate in bio-bleaching processes of paper industry. In addition, the features of active-site architecture of TfXyl10A in GH10 family were further analyzed. By mutating each residue at the -2 and -1 subsites to alanine, the binding force and enzyme activity of mutants were observably decreased. Interestingly, the mutant E51A, locating at the distal -3 subsite, exhibited 90% increase in relative activity compared with wild-type (WT) enzyme TfXyl10A-CD (the catalytic domain of TfXyl110A). This study explored the function of a GH10 xylanase containing a CBM2 domain and the contribution of amino acids in active-site architecture to catalytic activity. The results obtained provide guidance for the rational design of xylanases for industrial applications under high heat and alkali-based operating conditions, such as paper bleaching.

An R2R3-Type Transcription Factor OsMYBAS1 Regulates Seed Germination under Artificial Accelerated Aging in Transgenic Rice (Oryza sativa L.)

MYB-type transcription factors play an essential regulatory role in seed germination and the response to seedling establishment stress. This study isolated a rice R2R3-MYB transcription factor, OsMYBAS1, and functionally characterized its role in seed germination. There was no significant difference in the germination rate of each transgenic line in the standard germination test. However, compared to the germination rate of the wild type (WT) measured in the artificial accelerated aging test, the germination rates of the overexpression lines OE-OsMYBAS1-1 and OE-OsMYBAS1-2 were significantly increased by 25.0% and 21.7%, respectively. In contrast, the germination rates of the knockout mutants osmybas1-1 and osmybas1-2 were decreased by 21.7% and 33.3%, respectively. Additionally, the above data indicated that OsMYBAS1 possibly plays a positive role in rice seed germination. Moreover, the antioxidant enzyme activities of OsMYBAS1-overexpressing plants were enhanced by 38.5% to 151.0% while the superoxide dismutase (SOD) enzyme activity of osmybas1 mutants was decreased by 27.5%, and the malondialdehyde (MDA) content was increased by 24.7% on average. Interestingly, the expression of the antioxidation-related genes OsALDH3, OsAPX3, and OsCATC was enhanced in the OsMYBAS1 overexpression lines, which is consistent with the above results. Furthermore, transcriptome sequencing determined 284 differentially expressed genes (DEGs), which were mainly involved in the carbohydrate metabolic process, glycerolipid metabolism, and glycerophospholipid metabolism. Therefore, these findings provide valuable insight into the breeding of new rice varieties with high seed germination.

High-level production of the agmatine in engineered Corynebacterium crenatum with the inhibition-releasing arginine decarboxylase

BackgroundAgmatine is a member of biogenic amines and is an important medicine which is widely used to regulate body balance and neuroprotective effects. At present, the industrial production of agmatine mainly depends on the chemical method, but it is often accompanied by problems including cumbersome processes, harsh reaction conditions, toxic substances production and heavy environmental pollution. Therefore, to tackle the above issues, arginine decarboxylase was overexpressed heterologously and rationally designed in Corynebacterium crenatum to produce agmatine from glucose by one-step fermentation.ResultsIn this study, we report the development in the Generally Regarded as Safe (GRAS) l-arginine-overproducing C. crenatum for high-titer agmatine biosynthesis through overexpressing arginine decarboxylase based on metabolic engineering. Then, arginine decarboxylase was mutated to release feedback inhibition and improve catalytic activity. Subsequently, the specific enzyme activity and half-inhibitory concentration of I534D mutant were increased 35.7% and 48.1%, respectively. The agmatine production of the whole-cell bioconversion with AGM3 was increased by 19.3% than the AGM2. Finally, 45.26 g/L agmatine with the yield of 0.31 g/g glucose was achieved by one-step fermentation of the engineered C. crenatum with overexpression of speAI534D.ConclusionsThe engineered C. crenatum strain AGM3 in this work was proved as an efficient microbial cell factory for the industrial fermentative production of agmatine. Based on the insights from this work, further producing other valuable biochemicals derived from l-arginine by Corynebacterium crenatum is feasible.

Molecular Identification of Keratinase DgokerA from Deinococcus gobiensis for Feather Degradation

Keratin is a tough fibrous structural protein that is difficult to digest with pepsin and trypsin because of the presence of a large number of disulfide bonds. Keratin is widely found in agricultural waste. In recent years, especially, the development of the poultry industry has resulted in a large accumulation of feather keratin resources, which seriously pollute the environment. Keratinase can specifically attack disulfide bridges in keratin, converting them from complex to simplified forms. The keratinase thermal stability has drawn attention to various biotechnological industries. It is significant to identify keratinases and improve their thermostability from microorganism in extreme environments. In this study, the keratinases DgoKerA was identified in Deinococcus gobiensis I-0 from the Gobi desert. The amino acid sequence analysis revealed that DgoKerA was 58.68% identical to the keratinase MtaKerA from M. thermophila WR-220 and 40.94% identical to the classical BliKerA sequence from B. licheniformis PWD-1. In vitro enzyme activity analysis showed that DgoKerA exhibited an optimum temperature of 60 °C, an optimum pH of 7 and a specific enzyme activity of 51147 U/mg. DgoKerA can degrade intact feathers at 60 °C and has good potential for industrial applications. The molecular modification of DgoKerA was also carried out using site-directed mutagenesis, in which the mutant A350S enzyme activity was increased by nearly 30%, and the results provide a theoretical basis for the development and optimization of keratinase applications.

Loss of Function of Mutant IDS Due to Endoplasmic Reticulum-Associated Degradation: New Therapeutic Opportunities for Mucopolysaccharidosis Type II.

Mucopolysaccharidosis type II (MPS II) results from the dysfunction of a lysosomal enzyme, iduronate-2-sulfatase (IDS). Dysfunction of IDS triggers the lysosomal accumulation of its substrates, glycosaminoglycans, leading to mental retardation and systemic symptoms including skeletal deformities and valvular heart disease. Most patients with severe types of MPS II die before the age of 20. The administration of recombinant IDS and transplantation of hematopoietic stem cells are performed as therapies for MPS II. However, these therapies either cannot improve functions of the central nervous system or cause severe side effects, respectively. To date, 729 pathogenetic variants in the IDS gene have been reported. Most of these potentially cause misfolding of the encoded IDS protein. The misfolded IDS mutants accumulate in the endoplasmic reticulum (ER), followed by degradation via ER-associated degradation (ERAD). Inhibition of the ERAD pathway or refolding of IDS mutants by a molecular chaperone enables recovery of the lysosomal localization and enzyme activity of IDS mutants. In this review, we explain the IDS structure and mechanism of activation, and current findings about the mechanism of degradation-dependent loss of function caused by pathogenetic IDS mutation. We also provide a potential therapeutic approach for MPS II based on this loss-of-function mechanism.

Association between human paraoxonase 2 protein and efficacy of acetylcholinesterase inhibiting drugs used against Alzheimer's disease.

Serum Paraoxonase 2 (PON2) level is a potential biomarker owing to its association with a number of pathophysiological conditions such as atherosclerosis and cardiovascular disease. Since cholinergic deficiency is closely linked with Alzheimer’s disease (AD) progression, acetylcholinesterase inhibitors (AChEIs) are the treatment of choice for patients with AD. However, there is a heterogenous response to these drugs and mostly the subjects do not respond to the treatment. Gene polymorphism, the simultaneous occurrence of two or more discontinuous alleles in a population, could be one of the important factors for this. Hence, we hypothesized that PON2 and its polymorphic forms may be hydrolyzing the AChEIs differently, and thus, different patients respond differently. To investigate this, two AChEIs, donepezil hydrochloride (DHC) and pyridostigmine bromide (PB), were selected. Human PON2 wildtype gene and four mutants, two catalytic sites, and two polymorphic sites were cloned, recombinantly expressed, and purified for in vitro analysis. Enzyme activity and AChE activity were measured to quantitate the amount of DHC and PB hydrolyzed by the wildtype and the mutant proteins. Herein, PON2 esterase activity and AChE inhibitor efficiency were found to be inversely related. A significant difference in enzyme activity of the catalytic site mutants was observed as compared to the wildtype, and subsequent AChE activity showed that esterase activity of PON2 is responsible for the hydrolysis of DHC and PB. Interestingly, PON2 polymorphic site mutants showed increased esterase activity; therefore, this could be the reason for the ineffectiveness of the drugs. Thus, our data suggested that the esterase activity of PON2 was mainly responsible for the hydrolysis of AChEI, DHC, and PB, and that might be responsible for the variation in individual response to AChEI therapy.

Impact of high irradiance and UV-B on the photosynthetic activity, pro-/antioxidant balance and expression of light-activated genes in Arabidopsis thaliana hy4 mutants grown under blue light

The impacts of high-intensity light (HIL) (4 h) and UV-B radiation (1 h) on the photosynthetic activity, content of photosynthetic and UV-absorbing pigments (UAPs), activity of antioxidant enzymes (ascorbate peroxidase (APX) and guaiacol-dependent peroxidase (GPX)), content of thiobarbituric acid reactive substances (TBARs), expression of some light-regulated genes in 25-day-old wild type (WT) and the cryptochrome 1 (Cry1) hy4 mutant of A. thaliana Col-0 plants grown under blue light (BL) were studied. HIL and UV-B treatments led to decreases in the photosynthetic rate (Pn), photochemical activity of PSII (FV/FM) and PSII performance index (PIABS) of WT and mutant plants grown under high-intensity BL (HBL) and moderate intensity BL (MBL). However, in HBL plants, the decrease in the photosynthetic activity in hy4 plants was significantly greater than that in WT plants. In addition, hy4 HBL plants demonstrated lowered UAP and carotenoid contents as well as lower activity of APX and GPX enzymes. The difference in the decline in the photosynthetic activity of WT and hy4 plants grown at MBL in response to HIL was nonsignificant, while that in response to UV-B was small. We assume that the deficiency in cryptochrome 1 under HIL irradiation disrupts the interaction between HY5 and HFR1 transcription factors and photoreceptors, which affects the transcription of light-induced genes, such as CAB1, PSY and PAL1 linked to carotenoid and flavonoid biosynthesis. It was concluded that PA stress resistance in WT and hy4 plants depends on the light intensity and reduced stress resistance of hy4 at HBL, is likely linked to low UAP and carotenoid contents as well as lowered APX and GPX enzyme activities in hy4 mutants.

Directed Evolution Improves the Enzymatic Synthesis of L-5-Hydroxytryptophan by an Engineered Tryptophan Synthase.

L-5-Hydroxytryptophan is an important amino acid that is widely used in food and medicine. In this study, L-5-hydroxytryptophan was synthesized by a modified tryptophan synthase. A direct evolution strategy was applied to engineer tryptophan synthase from Escherichia coli to improve the efficiency of L-5-hydroxytryptophan synthesis. Tryptophan synthase was modified by error-prone PCR. A high-activity mutant enzyme (V231A/K382G) was obtained by a high-throughput screening method. The activity of mutant enzyme (V231A/K382G) is 3.79 times higher than that of its parent, and kcat/Km of the mutant enzyme (V231A/K382G) is 4.36 mM-1∙s-1. The mutant enzyme (V231A/K382G) reaction conditions for the production of L-5-hydroxytryptophan were 100 mmol/L L-serine at pH 8.5 and 35°C for 15 h, reaching a yield of L-5-hydroxytryptophan of 86.7%. Directed evolution is an effective strategy to increase the activity of tryptophan synthase.

Protein homology modeling‐informed active site characterization of a bacterial prostaglandin synthase ortholog from Nitrosomonas europaea

An open reading frame in the genome of Nitrosomonas europaea has been annotated as a prostaglandin H synthase (PGHS). We have expressed the protein, which demonstrates peroxidase activity with both hydrogen peroxide and lipid hydroperoxide substrates. Dioxygenase activity was not observed. In this study, we generate models of the N. europaea peroxidase to understand the structural explanation for its observed function. Using SWISS-MODEL, protein homology models were generated against PGHS, peroxidase, and dioxygenase templates. Using AutoDock Vina, the substrate 13(S)-hydroperoxy-(9Z, 11E)-octadecadienoic acid was inserted into the active site of each model to identify potential amino acids critical for activity. A conserved di-tyrosyl structure was observed in several unique models generated against redox enzyme templates. Amino acids in the active site predicted in the protein homology models were modified by site-directed mutagenesis. Specifically, the conserved tyrosyl structure was mutated to elucidate the importance of the structure in addition to the individual residues. Wild-type and mutant enzymes were expressed in BL21 cells and purified by Ni-NTA and Sephacryl S-200 chromatography. Kinetic assays using hydrogen peroxide and lipid hydroperoxide substrates are in progress to compare the mutant and wild-type enzyme activity. Identifying critical residues will contribute to our understanding of the evolutionary development of mammalian prostaglandin synthase.

Structural modifications on indole and pyrimidine rings of osimertinib lead to high selectivity towards L858R/T790M double mutant enzyme and potent antitumor activity

EGFR inhibitors represent a significant milestone for treatment of non-small cell lung cancer, however, they suffer from the acquired drug resistance. Utilizing osimertinib as the lead compound, this work has explored the structural modifications on the indole and pyrimidine rings of osimertinib to generate novel osimertinib derivatives. The in vitro enzymatic and cellular studies showed that the derivatives possessed high selectivity towards double mutant EGFR and potent antitumor activity. Particularly, compound 6b-1, the most active compound, exhibited excellent inhibitory activity against double mutant EGFR (IC50 = 0.18 nM) and wild-type EGFR (IC50 = 2.89 nM) as well as H1975 cells (IC50 = 1.44 nM). Western blot analysis showed that 6b-1 completely inhibited double mutant EGFR and Erk phosphorylation. In vivo test using xenograft model indicated that compound 6b-1 had better antitumor efficacy than osimertinib. More importantly, 6b-1 displayed many advantages in the pharmacokinetic study, including better oral bioavailability and metabolism character.

Effect of mutation at oxyanion hole residu (H110F) on activity of Lk4 lipase

Mutant of lipase at oxyanion hole (H110 F) was constructed. The gene was highly expressed in Eschericia coli BL21 (DE3) and the recombinant protein was purified using Ni-NTA affinity chromatography. The activity of mutant enzyme was significantly increased compared to that the wild type. Further comparison showed that both of the enzymes exhibited same optimum pH and temperature, and showed highest lipolytic activity on pNP-decanoate (C10). The wild type appeared lost of activity on C14 and C16 substrates meanwhile the mutant still showed activity up to 20 %. In the presence of non polar organic solvent such as n-hexane, the wild type became inactive enzyme meanwhile the mutant still remained 50 % of its activity. The results suggested that mutation at oxyanion hole (H110 F) caused enzyme-substrate interaction change resulting on elevation of activity, better activity toward longer carbon chain substrate and improving the activity in the present of non polar organic solvent.