Modulation of the Substrate Selectivity and Thermostability of Nitrile Hydratase Using Semi-rational Design Approach

Author:Cheng Zhong Yi

Supervisor:zhou zhe min


Degree Year:2019





Nitrile hydratase(NHase,EC a metalloenzyme found in both prokaryotic and eukaryotic organisms that catalyzes the formation of amides from nitriles.According to the metal ions contained,nitrile hydratase can be mainly classified into two types: cobalt-type nitrile hydratase(Co-NHase)and iron-type nitrile hydratase(Fe-NHase).Due to the promotion of the biochemical catalytic production of amide products,more and more attention has been focused on broadening the substrate spectrum of the enzyme,improving the catalytic selectivity and stability of this enzyme.Despite this,researches reported at this stage is still not prominent for the regioselectivity,stereoselectivity and enantioselectivity of most nitrile substrates,and most nitrile hydratases have poor thermal stability,which limits a wider range of applications of this enzyme.This paper,through semi-rational design which is based on bioinformatics and protein engineering,changed the regioselectivity and enantioselectivity of nitrile hydratase towards polycyanonitrile substrates and racemic nitrile substrates.The genen fusion strategy was also performed to improve the thermal stability of nitrile hydratase.The main findings are as follows:(1)Based on sequence alignment,the regioselectivity of nitrile hydratase towards polycyanonitrile substrates was altered by site-directed mutagenesis.The nitrile hydratase from Pseudomonas putida NRRL-18668(PpNHase)and Comomonas testosteroni(CtNHase)were used to study the catalytic characteristics towards nitriles with two cyano groups.It was found that PpNHase mainly catalyzes one cyano group in α,ω-dinitrile to form the intermediate ω-cyanomonoamide,while CtNHase catalyzes adiponitrile to form the final product α,ω-diamide.In such circumstance,both cyano groups of the substrate can be catalyzed.By aligning the primary amino acid sequences of PpNHase and CtNHase,the sequence similarity of the two was found to be 95.6%.There are 17 different amino acid between these two NHases,which are distributed on the two subunits,α and β.Using CtNHase as a template,the above 17 amino acids in PpNHase were mutated to amino acid residues at the corresponding sites of CtNHase,and the βL37F mutant among the 17 mutants was able to alter the regioselectivity of NHase towards the dinitrile substrates.The site was subjected to saturation mutagenesis,and finally three mutants,βL37F,βL37W and βL37Y,which mainly catalyze the production of α,ω-diamide from the dinitrile substrate were obtained.In addition,a site-directed saturation mutation was also carried out on the phenylalanine residue at position 37 of the β subunit of CtNHase,and two mutants mainly producing ω-cyanomonoamide,βF37L and βF37P,were obtained.Based on the comparison of the substrate access tunnel by Caver software,the larger the bottleneck of the substrate channel and the smaller the curvature,the nitrile hydratase tends to catalyze the two cyano groups in the dinitrile to form the diamide;the smaller the bottleneck of the substrate channel and the larger the curvature,the nitrile hydratase tends to catalyze only one of the cyano groups to form an ω-cyanomonoamide.(2)Based on molecular docking,the regioselectivity of nitrile hydratase for polycyanohydrin substrates was altered by site-directed mutagenesis.A protein model derived from low molecular weight nitrile hydratase(L-NHase)from Rhodococcus rhodochrous J1,was used to be docked with adiponitrile,malononitrile,phthalonitrile and terephthalonitrile determine to find out the possible binding amino acids in the substrate binding pocket.The single-point mutants βY68T and βW72Y were obtained by saturation mutagenesis.Their regioselectivity towards the dinitrile substrate changed from preference to diamide to preference to ω-cyanomonoamide.Further iterative saturation mutations resulted in a two-point combination mutant Y68T/W72 Y that produced only the ω-cyanomonoamide.Computer-aided analysis showed that the binding ability of the Y68T/W72 Y combinatorial mutation to the ω-cyanomonoamide product was weakened,and the binding free energy increased,resulting in the ω-cyanomonoamide not being further converted into diamide,and the regioselectivity was thus changed.(3)Based on steered molecular dynamics simulation,the enantioselectivity of the racemic nitrile substrate was altered by site-directed mutagenesis.L-NHase was used as the research object.By means of protein modeling and steered molecular dynamics simulation,four amino acid residues affecting the enantioselectivity of L-NHase towards racemic mandelonitrile were accurately located.The amino acid residue is subjected to a saturation mutation.The selectivity of the mutant strain for(S)-mandelonitrile was significantly improved,with the mutant βF37H showed an enantiomeric excess of 96.8%,which was much higher than 52.6% of the wild type.Steered molecular dynamics simulation analysis showed that the mutant increased the steric hindrance with(R)-mandelic nitrile,and the mutant βF37H was biased to select(S)-mandelic nitrile.Molecular dynamics simulation and semi-rational design reduce the workload of enzyme modification and effectively improve the efficiency of enzyme selectivity.(4)The stability of nitrile hydratase is improved based on protein fusion.L-NHase was used as a research object,and a highly heat-stable capping protein TERM was fused to the C-terminus of L-NHase to obtain a new fusion nitrile hydratase.The fusion nitrile hydratase still retained more than 80% residual enzyme activity after treatment at 50 ° C for 20 minutes,much higher than 40% of the wild type.Transmission electron microscopy showed that the fused nitrile hydratase could form spherical nanostructures with a diameter of about 25 nm.The results of protein modeling showed that the spherical structure might be a multi-subunit aggregate of L-NHase,and the formation of multimers was reduced.The specific surface area of the enzyme molecule is beneficial to the improvement of stability.