Protein and Peptide Letters - Volume 22, Issue 1, 2015

Acyltransferases (ATs) play an essential role in the polyketide biosynthesis through transferring acyl units into acyl carrier proteins (ACPs) via a self-acylation reaction and a transacylation reaction. Here we used AT10FkbA of FK506 biosynthetic polyketide synthase (PKS) from Streptomyces tsukubaensis YN06 as a model to study the specificity of ATs for acyl units. Our results show that AT10FkbA can form both malonyl-O-AT10FkbA and methylmalonyl-O-AT10FkbA in the self-acylation reaction, however, only malonyl-O-AT10FkbA but not methylmalonyl-O-AT10FkbA can transfer the acyl unit into ACPs in the transacylation reaction. Unlike some ATs that are known to control the acyl specificity in self-acylation reactions, AT10FkbA controls the acyl specificity in transacylation reactions.

Porins are integral membrane proteins found in the outer membrane of bacteria, mitochondria and chloroplasts. Herein, we have reviewed sequence and structural understanding about bacterial porins. The first porin structure from Rhodobacter capsulatus at 1.8 Å resolution in 1991 till the recent structural advancement, coupled by immunological properties, diffusion and ion permeation has been taken into account In the later part, we have presented our computational analysis of conformational mobility in selected porins. Atomic B-factors (in crystal structures) are indicative of the degree of intrinsic mobility associated with residues and secondary structural elements of a particular protein. We have explored and extended the intrinsic motilities within porins using selected six porins structures. These six porins were collected from PDB and B-factor analyses were performed using AWK scripts. Distributions of residues and mobilities were characteristic of different porins. These distribution patterns follow the level of homology at the sequence and structural level. The inner walls constituting the trimer interface were found to be more rigid than the outer walls. These mobility differences are intrinsic structural components of these porins.

Firefly luciferase is a relatively unstable protein and commonly loses its activity at room temperature because of structural changes. The structural and functional stability of this protein is critical for its enzymatic applications. Different approaches are applied to increase the stability of this enzyme such as designing of covalent cross-links (disulfide bonds). In this study, luciferase mutants containing one or two disulfide bonds were compared to the native protein for their fortheir structural, thermodynamic, and functional properties. Mutant forms of P. Pyralis luciferase A296C-A326C and A296C-A326C/P451C-V469C were used. Thermodynamic and biophysical studies were carried out using UV-Vis, fluorescence, circular dichroism, luminescence spectroscopy and differential scanning calorimetry (DSC). We observed that both mutant forms of the protein were more stable than the wild-type enzyme. However, the single disulfide bond containing mutant was structurally and functionally more stable than the mutant protein containing two disulfide bonds. Furthermore, the enzymatic activity of the single disulfide bond containing mutant protein was 7-folds greater than the wild type and the double disulfide bond proteins. The A296C-A326C mutation also increased the reversibility and disaggregation of the protein. The enhanced activity of the single disulfide bond mutant protein was contributed to the expansion of its active site cleft, which was confirmed by bioinformatics tools.

The human B12 trafficking chaperone protein hCblC is responsible for escorted delivery and early processing of B12 in intracellular B12 metabolism. In this study, we characterized a putative B12 trafficking chaperone of Caenorhabditis elegans (cCblC), which shows 26% amino acid sequence identity with hCblC. cCblC was shown to bind B12 with a broad specificity for the upper axial ligand, as previously observed with other homologous proteins. In addition, cCblC catalyzed glutathione (GSH)-dependent elimination of alkyl and GSH upper axial ligands from alkylcobalamins and glutathionylcobalamin (GSCbl), respectively. Dealkylation of methylcobalamin (MeCbl) generated cob(II)alamin with S-methylglutathione. Cob(I)alamin was detected as the intermediate for cob(II)alamin generation, indicating that the reaction is a nucleophilic displacement using the thiolate of GSH. Deglutathionylation of GSCbl also generated cob(II)alamin, via cob(I)alamin intermediate, with glutathione disulfide, indicating the reaction is chemically analogous with dealkylation. Cob(II)alamin generated by dealkylation and deglutathionylation was bound to cCblC in the base-off state and stable under aerobic conditions, which would be favorable for subsequent enzyme cofactor synthesis. These results demonstrate that cCblC is a B12 trafficking chaperone of C. elegans catalyzing dealkylation and deglutathionylation via a nucleophilic displacement using the thiolate of GSH.

The genome sequence analysis of Bacillus thuringiensis serovar konkukian S4 has shown to contain two chitinases (Chi74, Chi39) and two chitin-binding proteins (CBP50 and CBP24). The Chi74, Chi39 and CBP50 have been characterized previously. The chitin-binding protein CBP24 was cloned and heterologously expressed in Escherichia coli. The recombinant protein was purified using a Ni-NTA purification system. The purified protein was used to study its substrate binding activity using crystalline chitin variants as substrates. The Bmax and Kd values have shown that it preferably binds to β-type of the crystalline chitin at a range of pH with peak activity between 5.5-7.5. To elucidate the role of CBP24 in the chitin degradation system of S4, the purified chitinases Chi74, Chi39 along with the ChiA from Serratia proteamcualans were used in different combinations with the CBP24 and chitinolytic activity was assayed. It was shown that the CBP24 acts synergistically with chitin degradation activity of bacterial chitinases non-specifically. Moreover, the CBP24 has shown antifungal activity against plant pathogenic fungi Fusarium oxysporum and Rhizoctonia solani. The present study will lead us to develop a technology for environmental friendly conversion of chitin to valuable products.

The pH stability of lectin from black turtle bean was characterized by a variety of biophysical techniques over the pH range from 2.0 to 10.0. The zero-order and second-derivative UV spectra at different pH values indicated that the tertiary structure of the protein does not change significantly with changes in pH. The maxima emission fluorescence of the lectin upon excitation at 280 nm and 295 nm were found to be blue shifted 1.2 nm at pH 2.0, and with a red-shift of 1.0 nm at pH 10.0. The chemical denaturation results obtained by monitoring the intrinsic fluorescence of the lectin indicated that unfolding of the protein induced by guanidine hydrochloride (GdnHCl) could be described using a three-state model. Complete unfolding of the protein was observed at pH 7.2 in the presence of 6.5 M GdnHCl after 2 weeks, which suggested that the lectin was stable at various pHs. Irreversible thermal denaturation of lectin was also investigated at various pHs by differential scanning calorimetry (DSC). The first-order two-state kinetic model was applied to explain the scan-rate dependent DSC transitions. Both the higher activation energy (Ea) computed from the irreversible thermal denaturation and the three-state chemical denaturation process suggested that the lectin structure remained significantly unchanged from pH 2.0 to 10.0, which indicated that the structure of the lectin was stable over a wide pH range.

An inducible and aromatic nitrilase from Gordonia terrae was purified with a yield of 19%. The enzyme had turnover number of 63 s-1x 10-3, Km 1.4 mM and Vmax 95 Umg-1 protein for benzonitrile. The nitrilase of G. terrae was active at basic pH (7-10), moderate temperature (20-45 °C) and has a half-life of 4 h at 35 °C. MALDI analysis and amino acid sequence deduced from cloned nucleotide fragment showed 97% homology with putative amidohydrolase of Gordonia sputi NBRC 100414 and G. namibiensis. The enzyme showed regioselectivity towards hydroxybenzonitriles, as different position of hydroxyl group i.e. meta-, para- and orthosubstitutions on benzonitrile effect enzyme activity. The in-silico interactions of these substrates with the predicted 3D model of this enzyme also showed differential interaction between hydroxyl group of substrates and the polar amino acids surrounding enzyme’s active site. This leads to different proximity and orientation of substrates vis-a-vis their interaction with catalytic residues.

Elevated cadmium (Cd) concentrations in fishery byproducts are an environmental concern, that might be reduced by enzymatic removal and adsorption of the contaminants during recycling the byproducts as animal food. We cloned the gene for Arthrobacter nicotinovorans serine protease (ANISEP), which was isolated from the hepatopancreas of the Japanese scallop (Patiopecten yessoensis) and has been found to be an effective enzyme for Cd(II) removal. The gene is 993 bp in length and encodes 330 amino acids, including the pre (1–30) and pro (31–111) sequences. The catalytic triad consists of His, Asp, and Ser. Sequence similarities indicate that ANISEP is a extracellular serine protease. X-ray crystallography revealed structural similarities between ANISEP and the trypsin-like serine protease NAALP from Nesterenkonia sp. Site-directed mutagenesis identified Ser171 as catalytic residue. The keratinolytic activity of ANISEP was 10-fold greater than that of trypsin. ANISEP digested Cd(II)-bound recombinant metallothionein MT-10a from Laternula elliptica, but did not release Cd. These results further suggest ANISEP is a trypsin-like serine protease that can release Cd from the Japanese scallop hepatopancreas because of its strong keratinolytic activity.

Shrimp ovarian peritrophin (SOP), a major protein in jelly layer and cortical rods, plays a role in egg protection after spawning. Previous study, sequence of SOP gene from Fenneropenaeus merguiensis (Fm-SOP) was composed of domain A and domain B. The SOP domain A contains amino acid sequences between 1-80 of Fm-SOP. The domain A had six conserved cysteines which have been found in many antimicrobial peptides. The molecular weight of purified rSOP-A protein was about 9 kDa. The SOP domain B contains amino acid sequences 81-329 of Fm-SOP while SOP-B1 was amino acid sequence 182-275 of Fm-SOP. The molecular weight of purified rHis-SOP-B and rHis-SOP-B1 protein were about 38.5 and 18.0 kDa, respectively. Antimicrobial activities of rSOP-A, rHis-SOP-B and rHis-SOP-B1 protein were investigated by liquid growth inhibition assay. Minimal Inhibition Concentration (MIC) of rSOP-A against Staphylococcus aureus, Escherichia coli, Vibrio harveyi, Candida albicans and Fusarium oxysporum were 35, 280, 280, 570 and 15 μg/mL, respectively. The MIC of rHis-SOP-B against S. aureus, V. harveyi and F. oxysporum were 30, 270 and 500 μg/mL, respectively. And the MIC of rHis-SOP-B1 against S. aureus, V. harveyi and F. oxysporum were 20, 470 and 250 μg/mL, respectively. The rHis-SOP-B and rHis-SOP-B1 (1000 μg/mL) did not show antimicrobial activity against E. coli and C. albicans. Three purified proteins were able to agglutinate V. harveyi in vitro, displayed a chitinase activity and proteinase inhibition. In addition the stability of the proteins was tested and found decrease antimicrobial activity after incubation at 50 °C for 5 h.

Many proteins in chloroplast are regulated through the disulfide bond/thiol transformation to realize their activities. A homologue of VKOR (Vitamin K epoxide reductase) in Arabidopsis chloroplast is found to catalyze the disulfide bond formation in vivo and to mediate the specific phylloquinone reduction in vitro. It is also called LTO1 (Lumen Thiol Oxidoreductase 1). Investigations about functions and essential amino acid residues of AtVKOR have important theoretical significance to clarify the chloroplast redox regulation mechanism. In this study, several amino acids in the VKOR domain of AtVKOR were identified to be involved in binding of phylloquinone. Site-directed mutagenesis was used to study the function of these positions. The results suggested that residues Ser77, Leu87, Phe137 and Leu141 were quite important in the binding and catalyzing the reduction of phylloquinone. These residues were also involved in the electron transferring and disulfide bond formation of substrate proteins by motility assays in vivo, suggesting that the binding of phylloquinone not only affected the delivery of electrons to phylloquinone but also affected the whole electron transfer process. The conserved cysteines in the AtVKOR domain also played critical roles in phylloquinone reduction. When each of the four conserved cysteines was mutated to alanine, the mutants lost reduction activity entirely, suggesting that the four conserved cysteines played crucial roles in the electron transfer process.

Accumulation of ordered protein aggregates (or amyloids) represents a hallmark of many diseases (e.g., Alzheimer’s disease, type II diabetes, Parkinson’s diseases etc.), results from intermolecular association of partially unfolded proteins/ peptides. Such associations usually take place in highly crowded conditions. The aggregates, which are formed under in vitro and in vivo conditions exhibit substantial variations in their structure and function. Such heterogeneities in amyloids might arise due to macromolecular crowding that is usually omitted under in vitro conditions. The current study is an attempt to assess the effects of macromolecular crowding on amyloid formation using a model amyloidogenic peptide. The sequence of the peptide was derived from C-terminal region (RATQIPSYKKLIMY) of PAP(248-286), which naturally occurs in human semen as amyloid aggregates and is known for boosting HIV infectivity. This model peptide forms sedimentable and fibrillar aggregates in aqueous buffer and shows the characteristic features of amyloids. In the presence of macromolecular crowders the morphological features of the amyloids are significantly altered and resulted in the formation of shorter amyloid aggregates. The current study assumes the hypothesis that macromolecular crowding in the biological system favours formation of heterogeneous classes of aggregates and each of them might differ in their biophysical and biological properties.

G protein-coupled receptors (GPCRs) perform vital signaling functions and are involved in various diseases, making GPCRs major drug targets. GPCRs have seven α-helical transmembrane domains connected by three extracellular loops (ECLs) and three intracellular loops (ICLs). Among the three ICLs, ICL3 has been reported to have a critical function in interacting with downstream signaling molecules. Despite its important role in GPCR signaling, the structure of ICL3 has not been fully defined. In the present study, we used muscarinic acetylcholine receptor type 1 (M1) as a model system to analyze the structure of ICL3. Optimized purification conditions for M1_ICL3 comprised His-tag affinity purification and solubilization with n-dodecyl-b-D-maltopyranoside. Purified M1_ICL3 was analyzed using circular dichroism and hydrogen/deuterium exchange mass spectrometry; the results of these analyses suggested that M1_ICL3 is disordered and flexible.