Thermostability Improvement of L-Asparaginase from Acinetobacter soli via Consensus-Designed Cysteine Residue Substitution.

To extend the application range of L-asparaginase in food pre-processing, the thermostability improvement of the enzyme is essential. Herein, two non-conserved cysteine residues with easily oxidized free sulfhydryl groups, Cys8 and Cys283, of Acinetobacter soli L-asparaginase (AsA) were screened out via consensus design. After saturation mutagenesis and combinatorial mutation, the mutant C8Y/C283Q with highly improved thermostability was obtained with a half-life of 361.6 min at 40 °C, an over 34-fold increase compared with that of the wild-type. Its melting temperature (Tm) value reaches 62.3 °C, which is 7.1 °C higher than that of the wild-type. Molecular dynamics simulation and structure analysis revealed the formation of new hydrogen bonds of Gln283 and the aromatic interaction of Tyr8 formed with adjacent residues, resulting in enhanced thermostability. The improvement in the thermostability of L-asparaginase could efficiently enhance its effect on acrylamide inhibition; the contents of acrylamide in potato chips were efficiently reduced by 86.50% after a mutant C8Y/C283Q treatment, which was significantly higher than the 59.05% reduction after the AsA wild-type treatment. In addition, the investigation of the mechanism behind the enhanced thermostability of AsA could further direct the modification of L-asparaginases for expanding their clinical and industrial applications.

Evaluation of Activity Kinetic Parameters of SK319cys, As a New Cysteine Variant of Streptokinase: A Comparative Study.

BACKGROUND: Despite the extensive use of streptokinase in thrombolytic therapy, its administration may have some shortcomings like allergic reactions and relatively low half life. Specific PEGylation on cysteine at desired sites of streptokinase may alleviate these deficiencies and improve the quality of treatment. OBJECTIVE: This study was carried out to create a new cystein variant of streptokinase and compare its activity with formerly mutated SK263cys, SK45cys and intact streptokinase (Ski) to introduce superior candidates for specific PEGylation. METHOD: In silico study was carried out to select appropriate amino acid for cysteine substitution and accordingly mutagenesis was carried out by SOEing PCR. The mutated gene was cloned in E. coli, expressed, and purified by affinity chromatography. Activity of the purified proteins was assayed and kinetic parameters of enzymatic reaction were analyzed. RESULTS: According to in silico data, Arginine319 was selected for substitution with cysteine. SK319cys was achieved with 98% purity after cloning, expression and purification. It was shown that the enzymatic efficiency of SK319Cys and SK263cys was increased 18 and 21%, respectively, when compared to SKi (79.4 and 81.3 vs. 67.1µM-1min-1), while SK45cys showed 7% activity decrease (62.47µM-1min-1) compared to SKi. According to time-based activity assay, SK319Cys and SK263cys exhibited higher activity at lower substrate concentrations (100 and 200 µM), but at higher concentrations of substrate (400 and 800 µM), the proteins showed a very close trend of activity. CONCLUSION: SK319cys, as the new cysteine variant of streptokinase, together with SK263cys and SK45cys can be considered as appropriate molecules for specific PEGylation.

Dissecting the individual contribution of conserved cysteines to the redox regulation of RubisCO.

Oxidation of the cysteines from ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) leads to inactivation and promotes structural changes that increase the proteolytic sensitivity and membrane association propensity related to its catabolism. To uncover the individual role of the different cysteines, the sequential order of modification under increasing oxidative conditions was determined using chemical labeling and mass spectrometry. Besides, site-directed RubisCO mutants were obtained in Chlamydomonas reinhardtii replacing single conserved cysteines (Cys84, Cys172, Cys192, Cys247, Cys284, Cys427, Cys459 from the large and sCys41, sCys83 from the small subunit) and the redox properties of the mutant enzymes were determined. All mutants retained significant carboxylase activity and grew photoautotrophically, indicating that these conserved cysteines are not essential for catalysis. Cys84 played a noticeable structural role, its replacement producing a structurally altered enzyme. While Cys247, Cys284, and sCys83 were not affected by the redox environment, all other residues were oxidized using a disulfide/thiol ratio of around two, except for Cys172 whose oxidation was distinctly delayed. Remarkably, Cys192 and Cys427 were apparently protective, their absence leading to a premature oxidation of critical residues (Cys172 and Cys459). These cysteines integrate a regulatory network that modulates RubisCO activity and conformation in response to oxidative conditions.

Conformational changes during human P2X7 receptor activation examined by structural modelling and cysteine-based cross-linking studies.

The P2X7 receptor (P2X7R) is important in mediating a range of physiological functions and pathologies associated with tissue damage and inflammation and represents an attractive therapeutic target. However, in terms of their structure-function relationships, the mammalian P2X7Rs remain poorly characterised compared to some of their other P2XR counterparts. In this study, combining cysteine-based cross-linking and whole-cell patch-clamp recording, we examined six pairs of residues (A44/I331, D48/I331, I58/F311, S60/L320, I75/P177 and K81/V304) located in different parts of the extracellular and transmembrane domains of the human P2X7R. These residues are predicted to undergo substantial movement during the transition of the receptor ion channel from the closed to the open state, predictions which are made based on structural homology models generated from the crystal structures of the zebrafish P2X4R. Our results provide evidence that among the six pairs of cysteine mutants, D48C/I133C and K81C/V304C formed disulphide bonds that impaired the channel gating to support the notion that such conformational changes, particularly those in the outer ends of the transmembrane domains, are critical for human P2X7R activation.

A novel fibrillin-1 gene missense mutation associated with neonatal Marfan syndrome: a case report and review of the mutation spectrum.

BACKGROUND: Marfan syndrome (MFS) is a heritable disorder of connective tissue resulting from pathogenic variants of the fibrillin-1 gene (FBN1). Neonatal Marfan syndrome (nMFS) is rare and the most severe form of MFS, involving rapidly progressive cardiovascular dysfunction leading to death during early childhood. The constant enrichment of the nMFS mutation spectrum is helpful to improve our understanding of genotype-phenotype correlations in the disease. Herein, we report a novel dominant mutation in exon 26 of FBN1 (c.3331 T > C) in a sporadic case with nMFS. CASE PRESENTATION: An 8-month-old Han Chinese girl presented with the classic nMFS phenotype, including prominent manifestations of bone overgrowth, aortic root dilatation, and multiple cardiac valve dysfunctions. Genetic analysis revealed that she was heterozygous for a de novo FBN1 missense mutation (c.3331 T > C). The mutation leads to the substitution of a highly conserved FBN1 cysteine residue (p.Cys1111Arg), which is likely to severely perturb the FBN1 structure because of an alteration of the disulfide bond pattern in the calcium-binding epidermal growth factor-like (cbEGF) 12 domain. This variant was absent in 208 ethnically matched controls, providing further evidence that it may be causative of nMFS. An analysis of nMFS-associated mutations from the UMD-FBN1 database indicates that those de novo mutations altering disulfide bonds or Ca(2+) binding sites of the cbEGF domains encoded by exons 25-33, and a lack of phenotypic heterogeneity may be associated with an increased risk for nMFS. CONCLUSION: We diagnosed an infant with rare nMFS showing rapidly progressive cardiovascular dysfunction and widely systemic features. As the only causal FBN1 mutation identified in the patient, the missense mutation c.3331 T > C (p.Cys1111Arg) was associated with the severe phenotype of MFS. However, the pathogenicity of the novel mutation needs further confirmation in other patients with nMFS. Our review of the prominent characteristics of nMFS mutations relative to classic or incomplete MFS-related mutations will be helpful for the recognition of novel nMFS-associated variants.

Residues in the polar loop of subunit c in Escherichia coli ATP synthase function in gating proton transport to the cytoplasm.

Rotary catalysis in F1F0 ATP synthase is powered by proton translocation through the membrane-embedded F0 sector. Proton binding and release occur in the middle of the membrane at Asp-61 on the second transmembrane helix (TMH) of subunit c, which folds in a hairpin-like structure with two TMHs. Previously, the aqueous accessibility of Cys substitutions in the transmembrane regions of subunit c was probed by testing the inhibitory effects of Ag(+) or Cd(2+) on function, which revealed extensive aqueous access in the region around Asp-61 and on the half of TMH2 extending to the cytoplasm. In the current study, we surveyed the Ag(+) and Cd(2+) sensitivity of Cys substitutions in the loop of the helical hairpin and used a variety of assays to categorize the mechanisms by which Ag(+) or Cd(2+) chelation with the Cys thiolates caused inhibition. We identified two distinct metal-sensitive regions in the cytoplasmic loop where function was inhibited by different mechanisms. Metal binding to Cys substitutions in the N-terminal half of the loop resulted in an uncoupling of F1 from F0 with release of F1 from the membrane. In contrast, substitutions in the C-terminal half of the loop retained membrane-bound F1 after metal treatment. In several of these cases, inhibition was shown to be due to blockage of passive H(+) translocation through F0 as assayed with F0 reconstituted into liposomes. The results suggest that the C-terminal domain of the cytoplasmic loop may function in gating H(+) translocation to the cytoplasm.