Research Of Cobalt Incorporation Mechanism And Modification For Enhanced Thermostability Of Nitrile Hydratase In Pseudomonas Putida

Nitrile hydratase (NHase, EC4.2.1.84) is a metalloenzyme that can catalyze thehydration of a nitrile to the corresponding amide. It is widely used in the industrial application.Millions of acrylamide were produced by NHase. NHase catalysis possesses advantages suchas good selectivity, productivity, less side product, under common temperature and pressure,without product concentration and recycle of acrylnitrile and so on. A rapid improvement ofNHase catalysis was conducted in China.A wide range use of NHase induces its fundamental research. A long research of NHasecatalysis was conducted in our laboratory. Recently, a cobalt-containing NHase inRhodococcus rhodochrous J1, which uses a novel mode of post-translational maturation, wasdiscovered. This novel mode of post-translational maturation was called self-subunitswapping. This not only enhances the research of NHase catalysis but also raises theknowledge of people, which lays the foundation for new method. However, further study wasimperative.The mechanism of cobalt incorporation into NHase in Pseudomonas putidaNRRL-18668was discovered. The stability of NHase was also researched. The main resultswere conducted as follows:(1) P14K was successfully expressed in Escherichia coli. Nitrile hydratase (NHase)activators are essential for functional NHase biosynthesis. However, the activator P14K of thecobalt-containing NHase from P. putida is difficult to heterogeneously express in E. coli,which has retarded the clarification of the mechanism underlying the involvement of P14K inthe maturation of NHase. We here successfully expressed P14K through genetic modificationsaccording to N-end rule and analyze the mechanism underlying the instability of this protein.We found that mutation of the second N-terminal amino acid lysine to alanine or truncatingthe N-terminal16amino-acid sequence resulted in successful expression of P14K. Moreover,either addition of a strep tag alone (strep-P14K) or fusion of a pelB leader and srtep tagtogether (pelB-strep-P14K) at the N-terminus increased the expression of P14K.(2) The incorporation of cobalt into another type of Co-NHase, with a gene organizationof <α-subunit><β-subunit><activator protein>, was also dependent on self-subunit swapping.We successfully isolated a recombinant NHase activator protein (P14K) of P. putida. ThisP14K was found to form a complex α(P14K)2with the α-subunit of the NHase. Theincorporation of cobalt into the NHase of P. putida was confirmed to be dependent on theα-subunit substitution between cobalt-containing α(P14K)2and cobalt-free NHase. Theseresults expand on the general features of self-subunit swapping maturation..(3) The flexibility and positive charge of the C-terminal domain (C-domain) of theself-subunit swapping chaperone (P14K) of NHase from P. putida might play an importantrole in the cobalt incorporation for NHase activation. We first proposed a flexible C-domainfrom L85to A144using molecular dynamic simulation, and found that the C-domaintruncation and the P14K(G86I) mutation, which alter the C-domain flexibility, resulted invery low NHase activity. In addition, elimination of the positive charge in the C-domain also sharply affected NHase activity, and these mutants exhibited low cobalt content. Based on thestructural and energetic analyses, we proposed that the flexible, positively charged C-domainmost likely performs an external action that allows the cobalt-free NHase to overcome theenergetic barrier (203kcal×mol-1), resulting in a cobalt-containing NHase.(4) The thermo-stability of nitrile hydratase (NHase) was enhanced by fusing with two ofthe SAPs (EAK16and ELK16). When the ELK16was fused to the N-terminus of β-subunit,the resultant NHase (SAP-NHase-2) became an active inclusion body; EAK16fused NHasein the N-terminus of β-subunit (SAP-NHase-1) and ELK16fused NHase in the C-terminus ofβ-subunit (SAP-NHase-10) did not affect NHase solubility. Compared with the wild typeNHase, the thermal stability of SAP-NHase-1, SAP-NHase-2and SAP-NHase-10wereenhanced by45%,30%and50%, respectively.