We review major computational chemistry techniques applied in industrial enzyme studies, especially approaches intended for guiding enzyme engineering. These include molecular mechanics force field and molecular dynamics simulation, quantum mechanical and combined quantum mechanical/molecular mechanical approaches, electrostatic continuum models, molecular docking, etc. These approaches are essentially introduced from the following two angles for viewing: one is about the methods themselves, including the basic concepts, the primary computational results, and potential advantages and limitations; the other is about obtaining valuable information from the respective calculations to guide the design of mutants and mutant libraries.
Enzymes ; chemistry ; genetics ; metabolism ; Molecular Docking Simulation ; Molecular Dynamics Simulation ; Mutant Proteins ; chemistry ; genetics ; metabolism ; Protein Engineering ; Quantum Theory ; Static Electricity

Leucine-rich repeat kinase 2 (LRRK2) is a gene that, upon mutation, causes autosomal-dominant familial Parkinson's disease (PD). Yeast two-hybrid screening revealed that Snapin, a SNAP-25 (synaptosomal-associated protein-25) interacting protein, interacts with LRRK2. An in vitro kinase assay exhibited that Snapin is phosphorylated by LRRK2. A glutathione-S-transferase (GST) pull-down assay showed that LRRK2 may interact with Snapin via its Ras-of-complex (ROC) and N-terminal domains, with no significant difference on interaction of Snapin with LRRK2 wild type (WT) or its pathogenic mutants. Further analysis by mutation study revealed that Threonine 117 of Snapin is one of the sites phosphorylated by LRRK2. Furthermore, a Snapin T117D phosphomimetic mutant decreased its interaction with SNAP-25 in the GST pull-down assay. SNAP-25 is a component of the SNARE (Soluble NSF Attachment protein REceptor) complex and is critical for the exocytosis of synaptic vesicles. Incubation of rat brain lysate with recombinant Snapin T117D, but not WT, protein caused decreased interaction of synaptotagmin with the SNARE complex based on a co-immunoprecipitation assay. We further found that LRRK2-dependent phosphorylation of Snapin in the hippocampal neurons resulted in a decrease in the number of readily releasable vesicles and the extent of exocytotic release. Combined, these data suggest that LRRK2 may regulate neurotransmitter release via control of Snapin function by inhibitory phosphorylation.
Amino Acid Sequence ; Animals ; Exocytosis ; Female ; HEK293 Cells ; Humans ; Mice ; Molecular Sequence Data ; Mutant Proteins/metabolism ; Phosphorylation ; Phosphothreonine/metabolism ; Protein Binding ; Protein Interaction Mapping ; Protein Structure, Tertiary ; Protein-Serine-Threonine Kinases/*metabolism ; Qa-SNARE Proteins/metabolism ; Rats ; Rats, Sprague-Dawley ; Synaptosomal-Associated Protein 25/*metabolism ; Synaptotagmins/metabolism ; Vesicle-Associated Membrane Protein 2/metabolism ; Vesicular Transport Proteins/chemistry/*metabolism

Nine mutants (P2M1-9) were obtained using PCR with 5-BU based on DNA template (P2Y) encoding the active region of Parasporin-2. Mutant proteins were purified after expressing in E. coli BL21 cells, followed by assayed against hepatoma cells and normal liver cells by MTT. They showed diverse anti-hepatoma activities, in which two mutant proteins, P2M1 and P2M8, exhibited high cytotoxicity against hepatoma cell lines SMMC7721 and Be17402, meanwhile leaving normal liver cells Chang-liver unaffected. Structural comparison among P2Y, P2M1 and P2M8 showed that the length of beta-sheet or beta-fold, and the amount of alpha helix greatly affected the anti-hepatoma activity of Parasporin-2. Results based on amino acid alignment, molecular docking between P2Y, P2M1 or P2M8 and receptor, and mimic mutation demonstrated that amino acid residues at the sites of 52, 56, 58 and 208 on P2Y, especially the aromatic amino acids such as Trp, Phe, and Tyr were involved in the interactions.
Amino Acid Sequence ; Amino Acids, Aromatic ; biosynthesis ; genetics ; pharmacology ; Antineoplastic Agents ; pharmacology ; Carcinoma, Hepatocellular ; pathology ; Cell Line, Tumor ; Endotoxins ; chemistry ; pharmacology ; Escherichia coli ; genetics ; metabolism ; Humans ; Liver Neoplasms ; pathology ; Molecular Sequence Data ; Mutant Proteins ; pharmacology ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology

Bacterial Outer Membrane Proteins ; chemistry ; genetics ; metabolism ; Escherichia coli Proteins ; chemistry ; genetics ; metabolism ; Glutamic Acid ; genetics ; metabolism ; Histidine ; genetics ; Hydrogen-Ion Concentration ; Ion Channel Gating ; genetics ; Lipid Bilayers ; metabolism ; Mutant Proteins ; chemistry ; genetics ; metabolism ; Mutation ; Porins ; chemistry ; genetics ; metabolism

Mutations in LR RK2 (Leucine rich repeat kinase 2) are a major cause of Parkinson's disease (PD). We and others reported recently that expression of the pathogenic gainof-function mutant form of LRRK2, LRRK2 G2019S, induces mitochondrial fission in neurons through DLP1. Here we provide evidence that expression of LRRK2 G2019S stimulates mitochondria loss or mitophagy. We have characterized several LRRK2 interacting proteins and found that LRRK2 interacts with ULK1 which plays an essential role in autophagy. Knockdown of either ULK1 or DLP1 expression with shRNAs suppresses LRRK2 G2019S expression-induced mitochondrial clearance, suggesting that LRRK2 G2019S expression induces mitochondrial fission through DLP1 followed by mitophagy via an ULK1 dependent pathway. In addition to ULK1, we found that LRRK2 interacts with the endogenous MKK4/7, JIP3 and coordinates with them in the activation of JNK signaling. Interestingly, LRRK2 G2019S-induced loss of mitochondria can also be suppressed by 3 different JNK inhibitors, implying the involvement of the JNK pathway in the pathogenic mechanism of mutated LRRK2. Thus our findings may provide an insight into the complicated pathogenesis of PD as well as some clues to the development of novel therapeutic strategies.
Amino Acid Substitution ; Autophagosomes ; metabolism ; pathology ; Autophagy-Related Protein-1 Homolog ; chemistry ; genetics ; metabolism ; GTP Phosphohydrolases ; antagonists & inhibitors ; genetics ; metabolism ; Gene Knockdown Techniques ; HeLa Cells ; Humans ; Intracellular Signaling Peptides and Proteins ; chemistry ; genetics ; metabolism ; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 ; chemistry ; genetics ; metabolism ; MAP Kinase Signaling System ; Microtubule-Associated Proteins ; antagonists & inhibitors ; genetics ; metabolism ; Mitochondrial Degradation ; genetics ; physiology ; Mitochondrial Proteins ; antagonists & inhibitors ; genetics ; metabolism ; Mutant Proteins ; chemistry ; genetics ; metabolism ; Mutation ; Parkinson Disease ; genetics ; metabolism ; pathology ; Protein Interaction Domains and Motifs ; Recombinant Proteins ; chemistry ; genetics ; metabolism

Avian influenza A virus continues to pose a global threat with occasional H5N1 human infections, which is emphasized by a recent severe human infection caused by avian-origin H7N9 in China. Luckily these viruses do not transmit efficiently in human populations. With a few amino acid substitutions of the hemagglutinin H5 protein in the laboratory, two H5 mutants have been shown to obtain an air-borne transmission in a mammalian ferret model. Here in this study one of the mutant H5 proteins developed by Kawaoka's group (VN1203mut) was expressed in a baculovirus system and its receptor-binding properties were assessed. We herein show that the VN1203mut had a dramatically reduced binding affinity for the avian α2,3-linkage receptor compared to wild type but showed no detectable increase in affinity for the human α2,6-linkage receptor, using Surface Plasmon Resonance techonology. Further, the crystal structures of the VN1203mut and its complexes with either human or avian receptors demonstrate that the VN1203mut binds the human receptor in the same binding manner (cis conformation) as seen for the HAs of previously reported 1957 and 1968 pandemic influenza viruses. Our receptor binding and crystallographic data shown here further confirm that the ability to bind the avian receptor has to decrease for a higher human receptor binding affinity. As the Q226L substitution is shown important for obtaining human receptor binding, we suspect that the newly emerged H7N9 binds human receptor as H7 has a Q226L substitution.
Air Microbiology ; Crystallography, X-Ray ; Glycosylation ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; genetics ; metabolism ; Humans ; Influenza A Virus, H5N1 Subtype ; chemistry ; metabolism ; Influenza A Virus, H7N9 Subtype ; chemistry ; Models, Molecular ; Mutant Proteins ; chemistry ; genetics ; metabolism ; Protein Binding ; Protein Stability ; Receptors, Cell Surface ; genetics ; metabolism ; Solubility ; Surface Plasmon Resonance ; Temperature

The purpose of this paper is to establish sulfhydryl site-directed PEGylation method for lysostaphin and to evaluate effects of mutagenesis and modification of amino acid residue within putative linker on enzyme activity. On the basis of structural analysis of lysostaphin, amino acid 133-154 of tentative linker between the N-terminal and C-terminal domain were chosen as the candidate residues for site-directed mutagenesis to cysteine. Subsequently, sulfhydryl site-directed PEGylation was performed by reacting PEG-maleimide reagent with the newly introduced cysteine residue of the mutant lysostaphin. The Cys-mutant and PEG-modified proteins were both purified, and their enzymatic activity were further PEGylated lysostaphins. The mono-PEGylated lysostaphins were separated from unmodified lysostaphins through highly efficient one step method with Ni(2+)-NTA column chromatography. However, both Cys-mutant and PEGylated lysostaphin only retained partial activities of the wild-type enzyme. It suggests that sulfhydryl site-directed PEGylation modification of the tentative linker between the N-terminal and C-terminal domain may affect the catalytic activity of lysostaphin.
Anti-Infective Agents, Local ; chemistry ; metabolism ; Base Sequence ; Catalysis ; Cysteine ; chemistry ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Lysostaphin ; biosynthesis ; chemistry ; metabolism ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Mutant Proteins ; chemistry ; metabolism ; Polyethylene Glycols ; chemistry ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism ; Staphylococcus ; metabolism ; Sulfhydryl Reagents ; pharmacology

Arg-Gly-Asp (RGD)-toxin protein Lj-RGD3 of Lampetra japonica shares homologous with a Histidine-rich glycoprotein (HRG), and both RGD-toxin protein and HRG have antiangiogenic activities with different targets. To study the relationship between the function and the structure of Lj-RGD3, we studied the anti-angiogenic characteristics of both Lj-RGD3 and the mutation named Lj-112 of which three RGD motifs of Lj-RGD3 were deleted. We synthesized the gene of Lj-112, constructed it to the plasmid pET23b, and expressed the recombinant proteins in Escherichia coli BL21. Both recombinant proteins with the C-terminal his-tag were 15 kDa soluble proteins. Then we purified rLj-RGD3 and rLj-112 using the His-Bind affinity chromatography. To examine the effect of both proteins on bFGF-induced proliferation of ECV304 cell, we carried out the 3-(4,5)-dimethylthiahiazo (-z-yl)-3,5-di-phenytetrazoliumromide (MTT) assays. For cell migration and invasion assays, we used Transwell containing insert filter and Matrigel to imitate the in vivo environment. To examine whether both proteins were capable of interrupting the angiogenesis in vivo, we used the chick chicken embryonic chorioallantoic membrane (CAM) as an angiogenesis model. We used Integrin-linked kinase1 (ILK1) ELISA method to study functionary mechanisms of rLj-RGD3 and rLj-112. Both rLj-RGD3 and rLj-112 inhibited bFGF-induced proliferation of ECV304 cells in a dose-dependent manner with IC50 at 0.889 micromol/L and 0.160 micromol/L, respectively. The results of migration and invasion assays revealed that both rLj-RGD3 and rLj-112 showed significant inhibition on bFGF induced migration and invasion of ECV304; and rLj-112 was more active than rLj-RGD3. The result of CAM angiogenesis assay demonstrated that both proteins inhibited the angiogenesis in chick CAM, and rLj-112 was more active than rLj-RGD3. ELISA assay of ILK1 showed that both rLj-RGD3 and rLj-112 down-regulated ILK1 expression of ECV304 cell. The fact of rLj-112 was more active than rLj-RGD3 on anti-angiogenesis indicate that rLj-112 was likely with histidine-rich glycoprotein (HRG), and the factor of sequence homologous between rLj-RGD3 and HRG cannot enhance antiangiogenic activities of rLj-RGD3, the signal pathway of anti-angiogenesis of rLj-RGD3 and rLj-112 are differently.
Amino Acid Sequence ; Angiogenesis Inhibitors ; pharmacology ; Animals ; Base Sequence ; Cell Line ; Fish Venoms ; biosynthesis ; genetics ; isolation & purification ; pharmacology ; Humans ; Lampreys ; metabolism ; Molecular Sequence Data ; Mutant Proteins ; chemistry ; pharmacology ; Oligopeptides ; biosynthesis ; genetics ; isolation & purification ; pharmacology

The experiment was conducted by directed evolution strategy (error-prone PCR) to improve the activity of aflatoxin detoxifzyme with the high-throughput horse radish peroxidas and recessive brilliant green (HRP-RBG) screening system. We built up a mutant library to the order of 10(4). Two rounds of EP-PCR and HRP-RBG screening were used to obtain three optimum mutant strains A1773, A1476 and A2863. We found that mutant A1773 had upper temperature tolerance of 70 degrees C and that its enzyme activity was 6.5 times higher than that of the parent strain. Mutant strains A1476 worked well at pH 4.0 and its enzyme activity was 21 times higher than that of the parent strain. Mutant A2863 worked well at pH 4.0 and pH 7.5, and its enzyme activity was 12.6 times higher than that of the parent strain. With DNA sequencing we found that mutant A1773 revealed two amino acid substitutions, Glu127Lys and Gln613Arg. Mutant A1476 revealed four amino acid substitutions: Ser46Pro, Lys221Gln, Ile307Leu and Asn471lle. Mutant A2863 revealed four amino acid substitutions: Gly73Ser, Ile307Leu, Va1596Ala and Gln613Arg. The results provided a useful illustration for the deep understanding of the relationship between the function and structure of aflatoxin detoxifzyme.
Aflatoxin B1 ; antagonists & inhibitors ; chemistry ; Amino Acid Substitution ; Directed Molecular Evolution ; Enzyme Activation ; Enzyme Stability ; Multienzyme Complexes ; genetics ; metabolism ; Mutant Proteins ; genetics ; metabolism ; Point Mutation ; Polymerase Chain Reaction ; methods ; Protein Engineering

To improve the expression level and catalytic efficiency of (R)-carbonyl reductase from Candida parapsilosis in Escherichia coli, we optimized the mRNA secondary structure of (R)-carbonyl reductase gene in translation initiation region (from +1 to +78), and constructed the corresponding variant. The formation of hairpin structure was significantly reduced and the Gibbs free energy was dramatically decreased from -9.5 kcal/mol to -5.0 kcal/mol after optimization. As a result, the expression level of (R)-carbonyl reductase in the variant was increased by 4-5 times and its specific activity in cell-free extract was enhanced by 61.9% compared to the wild-type strain. When using the whole cells as catalyst and 2-hydroxyacetophenone as substrate with a high concentration of 5.0 g/L, the variant showed excellent performance to give (R)-1-phenyl-1, 2-ethanediol with optical purity of 93.1% enantiomeric excess and a yield of 81.8%, which were increased by 27.5% and 40.5% respectively than those of the wild-type. In conclusion, the optimization of mRNA secondary structure in translation initiation region can overcome the steric hindrance of translation startup, promote translation smoothly to acquire high expression of target protein, and favor protein folding correctly to efficiently improve the enzyme specific activity and biotransformation function.
Alcohol Oxidoreductases ; biosynthesis ; chemistry ; genetics ; Base Sequence ; Biocatalysis ; Candida ; enzymology ; Catalysis ; Escherichia coli ; genetics ; metabolism ; Molecular Sequence Data ; Mutant Proteins ; genetics ; Nucleic Acid Conformation ; Peptide Chain Initiation, Translational ; RNA, Messenger ; chemistry ; Recombinant Proteins ; biosynthesis ; genetics ; Stereoisomerism