A functional link between lariat debranching enzyme and the intron-binding complex is defective in non-photosensitive trichothiodystrophy.

The pre-mRNA life cycle requires intron processing; yet, how intron-processing defects influence splicing and gene expression is unclear. Here, we find that TTDN1/MPLKIP, which is encoded by a gene implicated in non-photosensitive trichothiodystrophy (NP-TTD), functionally links intron lariat processing to spliceosomal function. The conserved TTDN1 C-terminal region directly binds lariat debranching enzyme DBR1, whereas its N-terminal intrinsically disordered region (IDR) binds the intron-binding complex (IBC). TTDN1 loss, or a mutated IDR, causes significant intron lariat accumulation, as well as splicing and gene expression defects, mirroring phenotypes observed in NP-TTD patient cells. A Ttdn1-deficient mouse model recapitulates intron-processing defects and certain neurodevelopmental phenotypes seen in NP-TTD. Fusing DBR1 to the TTDN1 IDR is sufficient to recruit DBR1 to the IBC and circumvents the functional requirement for TTDN1. Collectively, our findings link RNA lariat processing with splicing outcomes by revealing the molecular function of TTDN1.

Improve Salinivibrio zinc-metalloprotease function in less polar organic solvents by increasing surface hydrophobicity.

Organic solvents tend to strip water from protein and thereby disrupt non-covalent forces and decrease enzyme activity and stability. In the present study, we have replaced the surface charge residues in Salinivibrio zinc-metalloprotease (SVP) with hydrophobic ones (E12V, D22I, D24A and D310I) in order to study the effects of surface hydrophobicity with hydrophobic strength of organic solvents. Compared to SVP, D24A exhibited an increase in kcat and catalytic efficiency and a reduction in thermal inactivation rate in aqueous solvent. Structural studies indicated that the replacement of surface charge residues with hydrophobic residues would not induce conformational changes. C50 value (the value of solvent concentration where 50% of enzyme activity remains), ki (irreversible thermoinactivation rate), and kinetic parameters of E12V, D22I, and D24A were higher in isopropanol and n-propanol. D24A is found to be the most efficient mutant for its remarkable decrease in ki value in the presence of isopropanol and n-propanol and a reduction in ki value in the presence of dimethylformamide (DMF) and methanol. C50 value in this variant was increased about 1.2% in DMF, 2% in methanol and isopropanol and 2.5% in n-propanol. Results revealed that, there was a correlation between surface hydrophobicity of SVP and hydrophobic strength of organic solvents.

Rational design toward developing a more efficient laccase: Catalytic efficiency and selectivity.

Laccases are multicopper oxidases that catalyze the oxidation of variety of substrates. The specificity and efficiency of laccases are clearly the important components leading to their remarkable uses. To develop an improved biocatalysts, site directed mutagenesis of laccase from Bacillus HR03 was carried out in the current study. Based on the ABTS-bound crystal structure of CotA from B. subtilis and alignment with closely related enzymes, T415 and T418 at the vicinity of the type 1 copper site were chosen and several mutants (T415I, T418I, T415G, T415G/T418I) were made. Kinetic parameters of the constructs were then determined using ABTS and SGZ as substrates. In comparison with the wild-type, catalytic efficiency toward ABTS was improved by 4 fold in T415I and 1.5 fold in T418I and T415G. T415I and T418I variants were identified to be almost 11 and 27 times more specific for ABTS than for SGZ compared with the wild type. T415I was also found to acquire enhanced thermal stability with the half-life of 60min at 80°C. Secondary and tertiary structure of mutants were analyzed by CD and fluorescence spectroscopy. Our result illustrated that replacement of residues in the substrate-binding pocket would change the specificity and efficiency of variants.