Integrative Structural and Computational Biology of Phytases for the Animal Feed Industry

Resistance to high temperature, acidic pH and proteolytic degradation during the pelleting process and in the digestive tract are important features of phytases as animal feed. The integration of insights from structural and in silico analyses into factors affecting thermostability, acid stability, proteolytic stability, catalytic efficiency and specific activity, as well as N-glycosylation, could improve the limitations of marginal stable biocatalysts with trade-offs between stability and activity. Synergistic mutations give additional benefits to single substitutions. Rigidifying the flexible loops or inter-molecular interactions by reinforcing non-bonded interactions or disulfide bonds, based on structural and roof mean square fluctuation (RMSF) analyses, are contributing factors to thermostability. Acid stability is normally achieved by targeting the vicinity residue at the active site or at the neighboring active site loop or the pocket edge adjacent to the active site. Extending the positively charged surface, altering protease cleavage sites and reducing the affinity of protease towards phytase are among the reported contributing factors to improving proteolytic stability. Remodeling the active site and removing steric hindrance could enhance phytase activity. N-glycosylation conferred improved thermostability, proteases degradation and pH activity. Hence, the integration of structural and computational biology paves the way to phytase tailoring to overcome the limitations of marginally stable phytases to be used in animal feeds.

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Improvement of thermostability and catalytic efficiency of glucoamylase from Talaromyces leycettanus JCM12802 via site-directed mutagenesis to enhance industrial saccharification applications

Biotechnology for Biofuels ◽

10.1186/s13068-021-02052-3 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Lige Tong ◽

Jie Zheng ◽

Xiao Wang ◽

Xiaolu Wang ◽

Huoqing Huang ◽

...

Keyword(s):

Disulfide Bonds ◽

Specific Activity ◽

Catalytic Efficiency ◽

Site Directed Mutagenesis ◽

Dynamics Simulation ◽

Optimal Temperature ◽

Correlation Matrices ◽

Catalytic Center ◽

Starch Industry ◽

Temperature Melting

Abstract Background Glucoamylase is an important industrial enzyme in the saccharification of starch into glucose. However, its poor thermostability and low catalytic efficiency limit its industrial saccharification applications. Therefore, improving these properties of glucoamylase is of great significance for saccharification in the starch industry. Results In this study, a novel glucoamylase-encoding gene TlGa15B from the thermophilic fungus Talaromyces leycettanus JCM12802 was cloned and expressed in Pichia pastoris. The optimal temperature and pH of recombinant TlGa15B were 65 ℃ and 4.5, respectively. TlGa15B exhibited excellent thermostability at 60 ℃. To further improve thermostability without losing catalytic efficiency, TlGa15B-GA1 and TlGa15B-GA2 were designed by introducing disulfide bonds and optimizing residual charge–charge interactions in a region distant from the catalytic center. Compared with TlGa15B, mutants showed improved optimal temperature, melting temperature, specific activity, and catalytic efficiency. The mechanism underlying these improvements was elucidated through molecular dynamics simulation and dynamics cross-correlation matrices analysis. Besides, the performance of TlGa15B-GA2 was the same as that of the commercial glucoamylase during saccharification. Conclusions We provide an effective strategy to simultaneously improve both thermostability and catalytic efficiency of glucoamylase. The excellent thermostability and high catalytic efficiency of TlGa15B-GA2 make it a good candidate for industrial saccharification applications.

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Integrating Terminal Truncation and Oligopeptide Fusion for a Novel Protein Engineering Strategy To Improve Specific Activity and Catalytic Efficiency: Alkaline α-Amylase as a Case Study

Applied and Environmental Microbiology ◽

10.1128/aem.02087-13 ◽

2013 ◽

Vol 79 (20) ◽

pp. 6429-6438 ◽

Cited By ~ 9

Author(s):

Haiquan Yang ◽

Long Liu ◽

Hyun-dong Shin ◽

Rachel R. Chen ◽

Jianghua Li ◽

...

Keyword(s):

Protein Engineering ◽

Active Site ◽

Specific Activity ◽

Catalytic Efficiency ◽

C Terminus ◽

N Terminus ◽

Engineering Strategy ◽

Specific Activities ◽

Novel Protein

ABSTRACTIn this work, we integrated terminal truncation and N-terminal oligopeptide fusion as a novel protein engineering strategy to improve specific activity and catalytic efficiency of alkaline α-amylase (AmyK) fromAlkalimonas amylolytica. First, the C terminus or N terminus of AmyK was partially truncated, yielding 12 truncated mutants, and then an oligopeptide (AEAEAKAKAEAEAKAK) was fused at the N terminus of the truncated AmyK, yielding another 12 truncation-fusion mutants. The specific activities of the truncation-fusion mutants AmyKΔC500-587::OP and AmyKΔC492-587::OP were 25.5- and 18.5-fold that of AmyK, respectively. Thekcat/Kmwas increased from 1.0 × 105liters · mol−1· s−1for AmyK to 30.6 × and 23.2 × 105liters · mol−1· s−1for AmyKΔC500-587::OP and AmyKΔC492-587::OP, respectively. Comparative analysis of structure models indicated that the higher flexibility around the active site may be the main reason for the improved catalytic efficiency. The proposed terminal truncation and oligopeptide fusion strategy may be effective to engineer other enzymes to improve specific activity and catalytic efficiency.

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Conformation-Specific Inhibitory Anti-MMP-7 Monoclonal Antibody Sensitizes Pancreatic Ductal Adenocarcinoma Cells to Chemotherapeutic Cell Kill

Cancers ◽

10.3390/cancers13071679 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1679

Author(s):

Vishnu Mohan ◽

Jean P. Gaffney ◽

Inna Solomonov ◽

Maxim Levin ◽

Mordehay Klepfish ◽

...

Keyword(s):

Monoclonal Antibody ◽

Active Site ◽

Drug Targets ◽

Fas Ligand ◽

Specific Activity ◽

Matrix Metalloproteases ◽

Ductal Adenocarcinoma ◽

Enzyme Active Site ◽

Induced Apoptosis

Matrix metalloproteases (MMPs) undergo post-translational modifications including pro-domain shedding. The activated forms of these enzymes are effective drug targets, but generating potent biological inhibitors against them remains challenging. We report the generation of anti-MMP-7 inhibitory monoclonal antibody (GSM-192), using an alternating immunization strategy with an active site mimicry antigen and the activated enzyme. Our protocol yielded highly selective anti-MMP-7 monoclonal antibody, which specifically inhibits MMP-7′s enzyme activity with high affinity (IC50 = 132 ± 10 nM). The atomic model of the MMP-7-GSM-192 Fab complex exhibited antibody binding to unique epitopes at the rim of the enzyme active site, sterically preventing entry of substrates into the catalytic cleft. In human PDAC biopsies, tissue staining with GSM-192 showed characteristic spatial distribution of activated MMP-7. Treatment with GSM-192 in vitro induced apoptosis via stabilization of cell surface Fas ligand and retarded cell migration. Co-treatment with GSM-192 and chemotherapeutics, gemcitabine and oxaliplatin elicited a synergistic effect. Our data illustrate the advantage of precisely targeting catalytic MMP-7 mediated disease specific activity.

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Enhancing the thermostability and activity of uronate dehydrogenase from Agrobacterium tumefaciens LBA4404 by semi-rational engineering

Bioresources and Bioprocessing ◽

10.1186/s40643-019-0267-3 ◽

2019 ◽

Vol 6 (1) ◽

Cited By ~ 1

Author(s):

Hui-Hui Su ◽

Fei Peng ◽

Pei Xu ◽

Xiao-Ling Wu ◽

Min-Hua Zong ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Rational Design ◽

Glucuronic Acid ◽

Specific Activity ◽

Value Added ◽

Catalytic Efficiency ◽

Site Directed Mutagenesis ◽

Glucaric Acid ◽

Triple Mutant ◽

Pharmaceutical Industries

Abstract Background Glucaric acid, one of the aldaric acids, has been declared a “top value-added chemical from biomass”, and is especially important in the food and pharmaceutical industries. Biocatalytic production of glucaric acid from glucuronic acid is more environmentally friendly, efficient and economical than chemical synthesis. Uronate dehydrogenases (UDHs) are the key enzymes for the preparation of glucaric acid in this way, but the poor thermostability and low activity of UDH limit its industrial application. Therefore, improving the thermostability and activity of UDH, for example by semi-rational design, is a major research goal. Results In the present work, three UDHs were obtained from different Agrobacterium tumefaciens strains. The three UDHs have an approximate molecular weight of 32 kDa and all contain typically conserved UDH motifs. All three UDHs showed optimal activity within a pH range of 6.0–8.5 and at a temperature of 30 °C, but the UDH from A. tumefaciens (At) LBA4404 had a better catalytic efficiency than the other two UDHs (800 vs 600 and 530 s−1 mM−1). To further boost the catalytic performance of the UDH from AtLBA4404, site-directed mutagenesis based on semi-rational design was carried out. An A39P/H99Y/H234K triple mutant showed a 400-fold improvement in half-life at 59 °C, a 5 °C improvement in $$ {\text{T}}_{ 5 0}^{ 1 0} $$ T 50 10 value and a 2.5-fold improvement in specific activity at 30 °C compared to wild-type UDH. Conclusions In this study, we successfully obtained a triple mutant (A39P/H99Y/H234K) with simultaneously enhanced activity and thermostability, which provides a novel alternative for the industrial production of glucaric acid from glucuronic acid.

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Kinetic studies and homology modeling of a dual-substrate linalool/nerolidol synthase from Plectranthus amboinicus

Scientific Reports ◽

10.1038/s41598-021-96524-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Nur Suhanawati Ashaari ◽

Mohd Hairul Ab. Rahim ◽

Suriana Sabri ◽

Kok Song Lai ◽

Adelene Ai-Lian Song ◽

...

Keyword(s):

Active Site ◽

Catalytic Efficiency ◽

Kinetic Studies ◽

Homology Model ◽

Biochemical Properties ◽

Terpene Synthase ◽

Monoterpene Synthase ◽

Terminal Domain ◽

Catalytic Active Site ◽

Plectranthus Amboinicus

AbstractLinalool and nerolidol are terpene alcohols that occur naturally in many aromatic plants and are commonly used in food and cosmetic industries as flavors and fragrances. In plants, linalool and nerolidol are biosynthesized as a result of respective linalool synthase and nerolidol synthase, or a single linalool/nerolidol synthase. In our previous work, we have isolated a linalool/nerolidol synthase (designated as PamTps1) from a local herbal plant, Plectranthus amboinicus, and successfully demonstrated the production of linalool and nerolidol in an Escherichia coli system. In this work, the biochemical properties of PamTps1 were analyzed, and its 3D homology model with the docking positions of its substrates, geranyl pyrophosphate (C10) and farnesyl pyrophosphate (C15) in the active site were constructed. PamTps1 exhibited the highest enzymatic activity at an optimal pH and temperature of 6.5 and 30 °C, respectively, and in the presence of 20 mM magnesium as a cofactor. The Michaelis–Menten constant (Km) and catalytic efficiency (kcat/Km) values of 16.72 ± 1.32 µM and 9.57 × 10–3 µM−1 s−1, respectively, showed that PamTps1 had a higher binding affinity and specificity for GPP instead of FPP as expected for a monoterpene synthase. The PamTps1 exhibits feature of a class I terpene synthase fold that made up of α-helices architecture with N-terminal domain and catalytic C-terminal domain. Nine aromatic residues (W268, Y272, Y299, F371, Y378, Y379, F447, Y517 and Y523) outlined the hydrophobic walls of the active site cavity, whilst residues from the RRx8W motif, RxR motif, H-α1 and J-K loops formed the active site lid that shielded the highly reactive carbocationic intermediates from the solvents. The dual substrates use by PamTps1 was hypothesized to be possible due to the architecture and residues lining the catalytic site that can accommodate larger substrate (FPP) as demonstrated by the protein modelling and docking analysis. This model serves as a first glimpse into the structural insights of the PamTps1 catalytic active site as a multi-substrate linalool/nerolidol synthase.

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Active-site serine mutants of the Streptomyces albus G β-lactamase

Biochemical Journal ◽

10.1042/bj2770647 ◽

1991 ◽

Vol 277 (3) ◽

pp. 647-652 ◽

Cited By ~ 10

Author(s):

F Jacob ◽

B Joris ◽

J M Frère

Keyword(s):

Active Site ◽

Specific Activity ◽

Site Directed Mutagenesis ◽

Beta Lactamase ◽

Wild Type ◽

Serine Residue ◽

Wild Type Enzyme ◽

Ph Values ◽

Streptomyces Albus ◽

Small Production

By using site-directed mutagenesis, the active-site serine residue of the Streptomyces albus G beta-lactamase was substituted by alanine and cysteine. Both mutant enzymes were produced in Streptomyces lividans and purified to homogeneity. The cysteine beta-lactamase exhibited a substrate-specificity profile distinct from that of the wild-type enzyme, and its kcat./Km values at pH 7 were never higher than 0.1% of that of the serine enzyme. Unlike the wild-type enzyme, the activity of the mutant increased at acidic pH values. Surprisingly, the alanine mutant exhibited a weak but specific activity for benzylpenicillin and ampicillin. In addition, a very small production of wild-type enzyme, probably due to mistranslation, was detected, but that activity could be selectively eliminated. Both mutant enzymes were nearly as thermostable as the wild-type.

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Protein hydrolysates from silkworm (Bombyx mori) pupae protein treated with a novel neutral protease

Journal of Insects as Food and Feed ◽

10.3920/jiff2021.0060 ◽

2021 ◽

pp. 1-18

Author(s):

R.A. Herman ◽

Z.-N. Li ◽

C. Xie ◽

J.-Z. Wang ◽

Y. Hu ◽

...

Keyword(s):

Bombyx Mori ◽

Water Solubility ◽

Specific Activity ◽

Catalytic Efficiency ◽

Apparent Molecular Weight ◽

Nutritional Composition ◽

Neutral Protease ◽

Degree Of Hydrolysis ◽

Silkworm Pupae ◽

Silkworm Bombyx Mori

Edible insects, regarded as a potential contributor to food security are currently given wide consideration due to their rich protein and other micronutrients contents. In this study, protease-assisted hydrolysis proposes an economically effective approach to hydrolyse proteins from silkworm (Bombyx mori) pupae to improve its functional properties. The proteolytic activity of a novel neutral protease (265.14 U/ml) with appreciable thermal activities, was identified using 16S rDNA as Stenotrophomonas maltophilia JW20 (SmNP20). The neutral protease with an apparent molecular weight of 28 kDa emerged active at pH 7 and maintained stability in pH range 6.0-8.0. The optimum temperature was 60 °C and stable at 55-60 °C, maintaining over 80% of its initial activity, with a half-life of 78.75, 89, 66.8 and 44 min at 50, 60, 70 and 80 °C. It was purified to 9.98-fold with a specific activity of 455.06 U/mg and 63.73% yield. The Km and Vmax values were 0.70 mg/ml and 9.48 μmol/min/mg, respectively. Enzymolysis with neutral protease enhanced the degree of hydrolysis (97.46±4.87%), increased water solubility over 50%, and a significant protein solubility of 63.44±0.65%. The Km and Vmax of the protein yield were 0.24 mg/ml and 165.63 μmol/min/mg respectively. A total of 17 amino acids have been detected in the hydrolysates obtained from the silkworm pupae protein. In comparison with neutrase and flavorzyme®, the enzyme possesses an elevated hydrolytic and catalytic efficiency. Emulsion activity and foam capacity ranged from 8-48 m2/g and 6-25% respectively. Hence, this study confirms the unique and efficient characteristics of an insect-enzyme correlation that is practically significant with potential improvement in nutritional composition and functional quality of insect proteins.

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Snake venom NAD glycohydrolases: primary structures, genomic location, and gene structure

PeerJ ◽

10.7717/peerj.6154 ◽

2019 ◽

Vol 7 ◽

pp. e6154 ◽

Cited By ~ 2

Author(s):

Ivan Koludarov ◽

Steven D. Aird

Keyword(s):

Snake Venom ◽

Active Site ◽

Disulfide Bonds ◽

Venom Gland ◽

Catalytic Residue ◽

Nad Glycohydrolase ◽

Adp Ribosyl Cyclase ◽

Genomic Location ◽

Cyclic Adp Ribose ◽

Very High

NAD glycohydrolase (EC 3.2.2.5) (NADase) sequences have been identified in 10 elapid and crotalid venom gland transcriptomes, eight of which are complete. These sequences show very high homology, but elapid and crotalid sequences also display consistent differences. As in Aplysia kurodai ADP-ribosyl cyclase and vertebrate CD38 genes, snake venom NADase genes comprise eight exons; however, in the Protobothrops mucrosquamatus genome, the sixth exon is sometimes not transcribed, yielding a shortened NADase mRNA that encodes all six disulfide bonds, but an active site that lacks the catalytic glutamate residue. The function of this shortened protein, if expressed, is unknown. While many vertebrate CD38s are multifunctional, liberating both ADP-ribose and small quantities of cyclic ADP-ribose (cADPR), snake venom CD38 homologs are dedicated NADases. They possess the invariant TLEDTL sequence (residues 144–149) that bounds the active site and the catalytic residue, Glu228. In addition, they possess a disulfide bond (Cys121–Cys202) that specifically prevents ADP-ribosyl cyclase activity in combination with Ile224, in lieu of phenylalanine, which is requisite for ADPR cyclases. In concert with venom phosphodiesterase and 5′-nucleotidase and their ecto-enzyme homologs in prey tissues, snake venom NADases comprise part of an envenomation strategy to liberate purine nucleosides, and particularly adenosine, in the prey, promoting prey immobilization via hypotension and paralysis.

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Identification of FDA-approved drugs as novel allosteric inhibitors of human executioner caspases

10.1101/356956 ◽

2018 ◽

Author(s):

R. N. V. Krishna Deepak ◽

Ahmad Abdullah ◽

Priti Talwar ◽

Hao Fan ◽

Palaniyandi Ravanan

Keyword(s):

Active Site ◽

Caspase 3 ◽

Specific Activity ◽

Special Importance ◽

Allosteric Inhibitors ◽

Dimerization Interface ◽

Executioner Caspases ◽

Approved Drugs ◽

Fda Approved Drugs

AbstractThe regulation of apoptosis is a tightly-coordinated process and caspases are its chief regulators. Of special importance are the executioner caspases, caspase-3/7, the activation of which irreversibly sets the cell on the path of death. Dysregulation of apoptosis, particularly an increased rate of cell death lies at the root of numerous human diseases. Although several peptide-based inhibitors targeting the homologous active site region of caspases have been developed, owing to their non-specific activity and poor pharmacological properties their use has largely been restricted. Thus, we sought to identify FDA-approved drugs that could be repurposed as novel allosteric inhibitors of caspase-3/7. In this study, we virtually screened a catalog of FDA-approved drugs targeting an allosteric pocket located at the dimerization interface of caspase-3/7. From among the top-scoring hits we short-listed five compounds for experimental validation. Our enzymatic assays using recombinant caspase-3 suggested that four out of the five drugs effectively inhibited caspase-3 enzymatic activity in vitro with IC50 values ranging ~10-55 μM. Structural analysis of the docking poses show the four compounds forming specific non-covalent interactions at the allosteric pocket suggesting that these molecules could disrupt the adjacently-located active site. In summary, we report the identification of four novel non-peptide allosteric inhibitors of caspase-3/7 from among FDA-approved drugs.

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Dynamism and Cooperativity Essential for Efficient Catalysis in Aspergillus Niger Monoamine Oxidase

10.26434/chemrxiv.7707857.v1 ◽

2019 ◽

Author(s):

Christian Curado-Carballada ◽

Ferran Feixas ◽

Sílvia Osuna

Keyword(s):

Aspergillus Niger ◽

Monoamine Oxidase ◽

Active Site ◽

Conformational Dynamics ◽

Catalytic Efficiency ◽

Building Blocks ◽

Chiral Amine ◽

Evolutionary Pathway ◽

Accelerated Molecular Dynamics ◽

Open States

Aspergillus niger Monoamine Oxidase (MAO-N) is a homodimeric enzyme responsible for the oxidation of amines into the corresponding imine. Laboratory evolved variants of MAO-N in combination with a non-selective chemical reductant represents a powerful strategy for the deracemisation of chiral amine mixtures and, thus, is of interest for obtaining chiral amine building blocks. MAO-N presents a rich conformational dynamics with a flexible ß-hairpin region that can adopt closed, partially closed and open states. Despite the ß-hairpin conformational dynamics is altered along the laboratory evolutionary pathway of MAO-N, the connection between the ß-hairpin conformational dynamics and active site catalysis still remains unclear. In this work, we use accelerated molecular dynamics to elucidate the potential interplay between the ß-hairpin conformational dynamics and catalytic activity in MAO-N wild type and its evolved D5 variant. Our study reveals a delicate communication between both MAO-N subunits that impacts the active site architecture, and thus its catalytic efficiency. In both MAO-N WT and the laboratory evolved D5 variant, the ß-hairpin conformation in one of the monomers affects the productive binding of the substrate in the active site of the other subunit. However, both MAO-N WT and D5 variants show a quite different behaviour due to the distal mutations introduced experimentally with Directed Evolution.

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