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

OBJECTIVETo study the over-expression of mutant p53 protein in non-mucinous adenocarcinoma in-situ (NMAIS) and invasive adenocarcinoma, NOS of lung.

METHODSImmunohistochemical study for p53 protein was performed on 17 cases of NMAIS and 70 cases of invasive adenocarcinoma, NOS of lung. The difference in p53 over-expression between the two tumor subtypes was analyzed.

RESULTSThe over-expression of mutant p53 protein was observed in 0 case (0%) of NMAIS and 37 cases (52.9%) of invasive adenocarcinoma, NOS of lung. The difference was of statistical significance (P = 0.000).

CONCLUSIONMutant p53 protein over-expression may play a role in the progression of NMAIS to invasive adenocarcinoma, NOS.

Adenocarcinoma ; metabolism ; Adenocarcinoma in Situ ; metabolism ; Humans ; Immunohistochemistry ; Mutant Proteins ; genetics ; metabolism ; Tumor Suppressor Protein p53 ; genetics ; metabolism

OBJECTIVETo study the correlation between IDH1 mutation, MGMT expression, clinicopathologic features and post-radiotherapy prognosis in patients with astrocytoma.

METHODSDetection of IDH1 mutation and MGMT expression was carried out in 48 cases of astrocytoma (WHO grade II to III) by EnVision method with immunohistochemical staining. Follow-up data, including treatment response and overall survival time, were analyzed.

RESULTSThe rates of IDH1 mutation and MGMT expression in astrocytomas were 62.7% (30/48) and 47.9% (23/48), respectively. There was a negative correlation between IDH1 mutation and MGMT expression (r = -0.641, P < 0.01). The age of patients with IDH1 mutation was younger at disease onset. The IDH1 mutation rate in patients with WHO grade II astrocytoma was higher than that in patients with WHO grade III tumor (P < 0.05). The age at onset was an independent factor affecting the expression of mutant IDH1. After radiotherapy, patients with IDH1 mutation+/MGMT- tumor carried a longer overall survival time than patients with IDH1 mutation-/MGMT+ tumor (P < 0.05).

CONCLUSIONSThere is a correlation between IDH1 mutation and MGMT expression in WHO grade II to III astrocytoma. Age at onset is an independent factor affecting the expression of mutant IDH1. Tumors with IDH1+/MGMT- pattern show better response to radiotherapy than tumors with IDH1-/MGMT+ pattern. Detection of IDH1 mutation and MGMT protein expression can provide some guidance in choice of treatment modalities in patients with astrocytoma.

Adult ; Age Factors ; Age of Onset ; Aged ; Astrocytoma ; genetics ; metabolism ; mortality ; pathology ; radiotherapy ; Brain Neoplasms ; genetics ; metabolism ; mortality ; pathology ; radiotherapy ; DNA Modification Methylases ; metabolism ; DNA Repair Enzymes ; metabolism ; Female ; Humans ; Isocitrate Dehydrogenase ; genetics ; Male ; Middle Aged ; Mutant Proteins ; genetics ; Mutation ; Prognosis ; Tumor Suppressor Proteins ; metabolism

OBJECTIVETo correlate the presence of chromosome 1p/19q deletion with the expression of R132H mutant IDH1 status in oligodendroglial tumors, and to explore molecular markers for predicting chemosensitivity of oligodendroglial tumors.

METHODSThe study included 75 oligodendroglial tumors (38 oligodendrogliomas and 37 oligoastrocytomas). Immunohistochemistry was used to detect the expression of R132H mutant IDH1 protein, and fluorescence in situ hybridization (FISH) was employed to detect 1p/19q deletion.

RESULTSDeletion of chromosome 1p and/or 19q was detected in 37 cases (37/75, 49.3%), among which co-deletion of 1p and 19q was seen in 34 cases (closely correlated, P < 0.01). Oligodendrogliomas WHOIIhad a slightly higher deletion rate than oligodendrogliomas WHO III, although without statistical significance. Oligodendrogliomas WHO IIand WHO III had a significantly higher deletion rate of chromosome 1p/19q than oligoastrocytomas WHO II and WHO III (P < 0.05). While combined loss of 1p/19q was always detected in oligodendrogliomas when FISH was positive, isolated 1p or 19q deletion was only found in oligoastrocytomas. The expression of R132H mutant IDH1 was detected in 51 of 75 cases (68.0%), in which oligodendrogliomas had a higher positive rate than oligoastrocytomas. Statistical analysis demonstrated a significant correlation between the expression of R132H mutant IDH1 protein and the presence of combined 1p/19q deletion in oligodendrogliomas (P < 0.05).

CONCLUSIONSA significant correlation was observed between the expression of R132H mutant protein and 1p/19q LOH.Expression of 132H mutant IDH1 protein is the potential biomarker for predicating the presence of 1p/19q deletion in oligodendrogliomas.

Aged ; Brain Neoplasms ; genetics ; metabolism ; Chromosome Deletion ; Chromosomes, Human, 19-20 ; genetics ; Chromosomes, Human, Pair 1 ; Humans ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Isocitrate Dehydrogenase ; genetics ; metabolism ; Middle Aged ; Mutant Proteins ; metabolism ; Neoplasm Proteins ; genetics ; metabolism ; Oligodendroglioma ; genetics ; metabolism

Efforts on directed evolution of sortase A to optimize its catalytic properties have been undertaken and shown the promise. To facilitate screening of sortase A mutants with expected catalytic properties, a novel ligation efficiency monitoring system, including reporter substrates GFP-LPETG and GGGYK-Biotin, was developed. GFP-LPETG, wild type sortase A, and a recently reported high activity sortase A mutant were prepared recombinantly from Escherichia coli BL21 (DE3). Taking advantage of the newly designed reporter system, the ligation efficiency catalyzed by wild type and mutant form of sortase A could be sensitively monitored via SDS-PAGE directly. Consistent with previous report, the mutant sortase A displayed much higher catalytic activity compared to wild type enzyme, indicating the new reporter system is easily and fast handled and sensitive. The application of this reporter system into systemic screening will facilitate future directed optimization of sortase A.
Aminoacyltransferases ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Biocatalysis ; Biotin ; Cysteine Endopeptidases ; genetics ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; Genes, Reporter ; Ligation ; Mutant Proteins ; genetics ; 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

We studied the mutation effect of subsites -3(Lys47), -7(146-152), and cyclization center (Tyr195) in active domain on product specificity of alpha-cyclodextrin glucanotransferase (alpha-CGTase) from Paenibacillus macerans sp. 602-1. The Lys47 was replaced by Thr47 and Tyr195 by Ile195, and the amino acids from 146 to 152 were replaced by Ile (named as delta6). All these mutant alpha-CGTases were actively expressed in E. coli BL21. Compared with the wild-type alpha-CGTase, the starch-degrading activities of all the mutant enzymes were declined. For mutant Y195I, the percentage of alpha-CD was decreased from 68% to 30%, and beta-CD was raised from 22.2% to 33.3%. Interestingly, gamma-CD was increased from 8.9% to 36.7% and became the main product, while the actual yield was increased from 0.4 g/L to 1.1 g/L. Mutant K47T and delta6 still produced alpha-CD as main product though the percentage of beta- and gamma-CD increased. Purified Y195I CGTase showed similar optimum temperature with the wild-type alpha-CGTase, but its optimum pH shifted from 5.0 to 6.0 with better pH stability. In summary, mutant Y195I CGTase has the potential to produce gamma-CD as the main product.
Escherichia coli ; genetics ; metabolism ; Glucosyltransferases ; genetics ; metabolism ; Mutant Proteins ; genetics ; metabolism ; Mutation ; Paenibacillus ; enzymology ; Recombinant Proteins ; genetics ; gamma-Cyclodextrins ; metabolism

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

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