Muhammad Akhtar

Mechanism of rhodopsin kinase regulation by recoverin.

Recoverin is suggested to inhibit rhodopsin kinase (GRK1) at high [Ca2+] in the dark state of the photoreceptor cell. Decreasing [Ca2+] terminates inhibition and facilitates phosphorylation of illuminated rhodopsin (Rh*). When recoverin formed a complex with GRK1, it did not interfere with the phosphorylation of a C‐terminal peptide of rhodopsin (S338‐A348) by GRK1. Furthermore, while GRK1 competed with transducin on interaction with rhodopsin and thereby suppressed GTPase activity of transducin, recoverin in the complex with GRK1 did not influence this competition. Constructs of GRK1 that encompass its N‐terminal, catalytic or C‐terminal domains were used in pull‐down assays and surface plasmon resonance analysis to monitor interaction. Ca2+‐recoverin bound to the N‐terminus of GRK1, but did not bind to the other constructs. GRK1 interacted with rhodopsin also by its N‐terminus in a light‐dependent manner. No interaction was observed with the C‐terminus. We conclude that inhibition of GRK1 by recoverin is not the result of their direct competition for the same docking site on Rh*, although the interaction sites of GRK1/Rh* and GRK1/recoverin partially overlap. The N‐terminus of GRK1 is recognized by Rh* leading to a conformational change which moves the C‐terminus of Rh* into the catalytic kinase groove. Ca2+‐recoverin interacting with the N‐terminus of GRK1 prevents this conformational change and thus blocks Rh* phosphorylation by GRK1.

Production of bubaline somatotropin by auto-induction in Escherichia coli

Production of His–BbST [hexahistidine‐tagged BbST (bubaline somatotropin)] by auto‐induction in high‐density shake‐flask cultures coupled with a single‐step, on‐column purification and refolding strategy is described here. To optimize expression of BbST, different media and expression conditions were tested. The highest expression levels of BbST, exceeding 30% of the total Escherichia coli cellular proteins, were achieved when YNG and M9NG media were used. Using these auto‐inducing media, the final concentration of BbST increased up to 455 mg/l and was severalfold higher than that obtained by isopropyl β‐d‐thiogalactoside induction. Most of the target protein, however, was in the form of inclusion bodies, which were solubilized in 8 M urea solution (pH 8.5). Using immobilized‐metal‐ion‐affinity chromatography, His–BbST could be purified from solubilized sample to >97% homogeneity in a single step in a biologically active state as judged by its efficient growth‐promoting activity in Nb2 rat lymphoma cell proliferation assays. The expression and purification scheme, presented here, has a potential of scaling up to obtain pure and biologically active His–BbST relatively inexpensively for further studies and applications.

The nature of the carbohydrate binding module determines the catalytic efficiency of xylanase Z of Clostridium thermocellum.

Xylanase Z of Clostridium thermocellum exists as a complex in the cellulosome with N-terminus feruloyl esterase, a carbohydrate binding module (CBM6) and a dockerin domain. To study the role of the binding modules on the activity of XynZ, different variants with the CBM6 attached to the catalytic domain at its C-terminal (XynZ-CB) and N-terminal (XynZ-BC), and the CBM22 attached at N-terminus (XynZ-BC) were expressed in Escherichia coli at levels around 30% of the total cell proteins. The activities of XynZ-BC, XynZ-CB and XynZ-BC were 4200, 4180 and 20,700U μM(-1) against birchwood xylan, respectively. Substrate binding studies showed that in case of XynZ-BC and XynZ-CB the substrate birchwood xylan remaining unbound were 51 and 52%, respectively, whereas in the case of XynZ-BC the substrate remaining unbound was 39% under the assay conditions used. The molecular docking studies showed that the binding site of CBM22 in XynZ-BC is more exposed and thus available for substrate binding as compared to the tunnel shape binding pocket produced in XynZ-BC and thus hindering the substrate binding. The substrate binding data for the two constructs are in agreement with this explanation.

Enhanced production and characterization of a β-glucosidase from Bacillus halodurans expressed in Escherichia coli

A putative β-glucosidase gene from the genome of Bacillus halodurans C-125 was expressed in E. coli under the regulation of T7lac promoter. On induction with isopropyl-β-D-1-thiogalactopyranoside, the enzyme expressed at ∼40% of the cell protein producing 238 mg/liter culture. With increase in culture cell density to A600 12 in auto-inducing M9NG medium, β-glucosidase production increased 3-fold. Approximately 70% of the expressed enzyme was in a soluble form, while the rest was in an insoluble fraction of the cell lysate. The soluble and active form of the expressed enzyme was purified by ammonium sulfate precipitation followed by ion-exchange chromatography to a purity >98%. The mass of the enzyme as determined by MALDI-TOF mass spectrometry was 51,601 Da, which is nearly the same as the calculated value. Phylogenetic analysis of the β-glucosidase of B. halodurans was found to cluster with members of the genus Bacillus. Temperature and pH optima of the enzyme were found to be 45°C and 8.0, respectively, under the assay conditions. Km and kcat against p-nitrophenyl-β-D-glucopyranoside were 4 mM and 0.75 sec−1, respectively. To our knowledge, this is the first report of high-level expression and characterization of a β-glucosidase from B. halodurans.

The Reverse Gyrase from Pyrobaculum calidifontis, a Novel Extremely Thermophilic DNA Topoisomerase Endowed with DNA Unwinding and Annealing Activities

Background: The thermophilic DNA topoisomerase reverse gyrase induces DNA-positive supercoiling. Results: The novel reverse gyrase, PcalRG, is presented. Conclusion: PcalRG is the most efficient and robust reverse gyrase known, and the first inducing ATP-dependent unwinding of Holliday junctions and annealing of single-stranded oligonucleotides. Significance: PcalRG shares structural and functional features with evolutionary conserved helicase-topoisomerase complexes involved in genome stability. Reverse gyrase is a DNA topoisomerase specific for hyperthermophilic bacteria and archaea. It catalyzes the peculiar ATP-dependent DNA-positive supercoiling reaction and might be involved in the physiological adaptation to high growth temperature. Reverse gyrase comprises an N-terminal ATPase and a C-terminal topoisomerase domain, which cooperate in enzyme activity, but details of its mechanism of action are still not clear. We present here a functional characterization of PcalRG, a novel reverse gyrase from the archaeon Pyrobaculum calidifontis. PcalRG is the most robust and processive reverse gyrase known to date; it is active over a wide range of conditions, including temperature, ionic strength, and ATP concentration. Moreover, it holds a strong ATP-inhibited DNA cleavage activity. Most important, PcalRG is able to induce ATP-dependent unwinding of synthetic Holliday junctions and ATP-stimulated annealing of unconstrained single-stranded oligonucleotides. Combined DNA unwinding and annealing activities are typical of certain helicases, but until now were shown for no other reverse gyrase. Our results suggest for the first time that a reverse gyrase shares not only structural but also functional features with evolutionary conserved helicase-topoisomerase complexes involved in genome stability.

Influence of positioning of carbohydrate binding module on the activity of endoglucanase CelA of Clostridium thermocellum.

This study reports characteristics of different derivatives produced between CelA, a major endoglucanase of Clostridium thermocellum and carbohydrate binding domain of family 3a (CBM3a). In addition to the native form of the endoglucanase containing catalytic and dockerin domains (CelA-CD), its derivatives consisting of catalytic domain without dockerin domain (CelA-C), catalytic domain linked with the binding domain at N-, C- and both termini (CelA-BC, CelA-CB and CelA-BCB, respectively), two catalytic domains cloned in tandem (CelA-CC) and two catalytic domains intervened by a binding domain (CelA-CBC) were expressed in Escherichia coli at levels of 40, 43, 28, 30, 20, 20 and 10%, respectively of the total cell proteins. Specific activities of CelA-CD, CelA-C, CelA-BC, CelA-CB, CelA-CC, CelA-BCB and CelA-CBC against carboxymethyl cellulose (CMC) were 8.1, 7.0, 12.1, 8.5, 11.8, 10.2 and 23.5Umg(-1) enzyme while activities against pre-treated bagasse were 490, 250, 1400, 600, 810, 710 and 2270μmoles reducing sugars released per μmole of the enzyme, respectively, under the assay conditions used. Thus the activities of CelA-BC and CelA-CBC showed nearly 3- and 5-fold increase against pre-treated bagasse as compared to that of the native form of the enzyme, CelA-CD. Molecular modeling studies using MODELLER show that the binding residues of CBM3a and the active site residues of the catalytic domain are more favorably oriented for binding and hydrolysis of the polysaccharide in the case of CelA-BC as compared to those in CelA-CB, which corresponds with higher activity of the former.

Studies on the regioselectivity and kinetics of the action of trypsin on proinsulin and its derivatives using mass spectrometry.

Human M-proinsulin was cleaved by trypsin at the R(31)R(32)-E(33) and K(64)R(65)-G(66) bonds (B/C and C/A junctions), showing the same cleavage specificity as exhibited by prohormone convertases 1 and 2 respectively. Buffalo/bovine M-proinsulin was also cleaved by trypsin at the K(59)R(60)-G(61) bond but at the B/C junction cleavage occurred at the R(31)R(32)-E(33) as well as the R(31)-R(32)E(33) bond. Thus, the human isoform in the native state, with a 31 residue connecting C-peptide, seems to have a unique structure around the B/C and C/A junctions and cleavage at these sites is predominantly governed by the structure of the proinsulin itself. In the case of both the proinsulin species the cleavage at the B/C junction was preferred (65%) over that at the C/A junction (35%) supporting the earlier suggestion of the presence of some form of secondary structure at the C/A junction. Proinsulin and its derivatives, as natural substrates for trypsin, were used and mass spectrometric analysis showed that the k(cat.)/K(m) values for the cleavage were most favourable for the scission of the bonds at the two junctions (1.02±0.08×10(5)s(-1)M(-1)) and the cleavage of the K(29)-T(30) bond of M-insulin-RR (1.3±0.07×10(5)s(-1)M(-1)). However, the K(29)-T(30) bond in M-insulin, insulin as well as M-proinsulin was shielded from attack by trypsin (k(cat.)/K(m) values around 1000s(-1)M(-1)). Hence, as the biosynthetic path follows the sequence; proinsulin→insulin-RR→insulin, the K(29)-T(30) bond becomes shielded, exposed then shielded again respectively.

Expression enhancement of bubaline somatotropin in E. coli through gene modifications in the 5′-end coding region

This study addresses the problem of poor expression of somatotropin (ST) gene in E. coli and describes expression enhancement through silent and non-silent gene modifications. A series of constructs with codon optimization, substitution, deletion or addition in the 5-region of the sequence encoding bubaline ST (BbST) were prepared. In the native form, the BbST expression was barely discernible on SDS-gel of the total E. coli cellular proteins (TCP). Introduction of silent and non-silent mutations in +2 to +8 codons, however, raised the expression levels to varying extents. In some constructs, a single base variation, i.e., G-->A or G-->C led to a remarkable increase in BbST expression (up to 28% of the TCP) whereas in the case of G-->T substitution the expression dropped to undetectable levels. Deletion of native GCC codon and addition of CAUCAC repeat thrice at +2 position enhanced the expression up to 48%, while insertion of NGG codons at the same position caused just a modest increase in expression. Differences in expression appeared as if related to the nature of early downstream codons (especially +2) and the stability of mRNA secondary structure although the levels of intracellular mRNA pools, as analyzed by real-time RT-PCR were quite similar. Overall, the study highlights the importance of 5-end codon adaptations in solving the problems encountered in expressing the eukaryotic genes in E. coli.

Characterization of a β-xylosidase produced by a mutant derivative of Humicola lanuginosa in solid state fermentation

The production of extracellular β-xylosidase by a newly isolated mutant derivative of Humicola lanuginosa M7D on lignocellulosic substrates and xylan was maximized in solid state fermentation by adopting a search technique varying one parameter at a time. The H. lanuginosa mutant achieved maximum production of β-xylosidase (728xa0IUxa0g−1 substrate, YP/S) when grown on Vogel’s medium containing xylan, followed by medium containing corncobs (669xa0IUxa0g−1) supplemented with corn steep liquor (initial pHxa06.5, moisture level 75%) at 45°C. Purified mutant- and parent-derived enzyme exhibited Km values of 1.8 and 2.0xa0mM, respectively. Both enzymes were optimally active at pHxa08.5 and a temperature of 60°C. Both enzymes displayed high thermostability, with a half-life of 2.9 and 0.9xa0min, enthalpy of denaturation (ΔH*) of 102.1 and 110.10xa0kJxa0mol−1, entropy of denaturation (ΔS*) of −38.5 and −4.5xa0J/molxa0K, and free energy of denaturation (ΔG*) of 115.7 and 111.7xa0kJ/mol at 80°C for the mutant- and parent organism-derived enzymes, respectively. The kinetic and thermodynamic properties suggest that the β-xylosidases from both strains are superior to several previously reported enzymes from other thermophilic species, and may have potential applications in various industrial fields.

Structural evidence for the partially oxidized dipyrromethene and dipyrromethanone forms of the cofactor of porphobilinogen deaminase: structures of the Bacillus megaterium enzyme at near-atomic resolution

The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses a key early step in the biosynthesis of tetrapyrroles in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. Two near-atomic resolution structures of PBGD from B. megaterium are reported that demonstrate the time-dependent accumulation of partially oxidized forms of the cofactor, including one that possesses a tetrahedral C atom in the terminal pyrrole ring.