Structure And Function Analysis Of Low-molecular-weight Nitrile Hydratase Activator From Rhodococcus Rhodochrous J1

Nitrile hydratase?NHase,EC 4.2.1.84?is a kind of metalloenzyme capable of catalyzing the hydration of nitrile compounds to amide compounds.The active unit of NHase is composed of an?subunit and a?subunit.According to the types of the metal ion in the active center,NHases are divided into iron-type nitrile hydratase?Fe-NHase?and cobalt-type nitrile hydratase?Co-NHase?.Activators,which assist nitrile hydratases to take up metal ions and to be posttranslationally modified,are essential for NHase biosynthesis.Previous studies showed that the maturation of low-molecular-weight nitrile hydratase?L-NHase?from Rhodococcus rhodochrous J1 used a“self-subunit swapping”mechanism:The?subunits exchange occurred between the cobalt-free apoenzyme apo-?₂?₂ and the cobalt-containing protein complex holo-?e₂ to form the active holoenzyme holo-?₂?₂.The activator e and other Co-NHase activators participated in the“self-subunit swapping”process play a key role in the post-translational maturation of Co-NHase,however,there are few related researches about them and their action mechanisms have not yet been clarified.This study analyzed the structures and functions of activator e and several other Co-NHase activators through the methods of molecular biology,structural biology and bioinformatics.The main results are as follows:?1?Structural biology analysis of L-NHase?holo-?₂?₂?and the protein complex holo-?e₂.Through the efficient heterologous expression and the protein purification process including ammonium sulfate precipitation,anion exchange chromatography,affinity chromatography,and gel filtration chromatography,L-NHase and holo-?e₂ with high purity and high concentration were obtained.The screening and optimization of protein crystallization conditions were further performed by the sessile drop vapor diffusion method,and the L-NHase crystals with high qualities were successfully prepared under conditions of 70mg·m L^-1 purified enzyme,0.1 M BIS-TRIS propane?pH=9.0?,and 35%?W/V?PEG 6000.The diffraction data of the L-NHase crystal with a resolution of about 3?was obtained by X-ray diffraction.?2?Stability analysis of the protein complex holo-?e₂ and the activator e.The stability of the purified holo-?e₂ was tested.It was found that after incubation at 4?for 4 d,the activatore spontaneously degraded,meanwhile,it led to the cleavage of the?subunit.It was found further that the degradation of the activator e and the cleavage of the?subunit could be inhibited by broad-spectrum protease inhibitors.The stability of the purified activator e was also tested,and the similar spontaneous degradation was observed,indicating that the activator e was unstable and it might have protease activities.?3?Structure and function analysis of several Co-NHase activators.Four heterologous Co-NHase activators?g,p,c,and a?were coexpressed with L-NHase respectively,and they were found to be able to activate L-NHase by assisting?subunits to take up cobalt ions.The activation ability of the activator a was similar to that of the activator e,while the activation of the activator g was the weakest.Through sequence analysis and structural simulation,it was found that five activators?e,g,p,c,and a?all had the conserved domains TIGR03889,which had similarities with the N-terminal sequence of the?subunit to some extent,and the differences of N-terminal structures and C-terminal structures between the activator e and the activator g were observed.Replacing the N-terminal sequence of the activator g with the N-terminal sequence of the activator e,and adding the C-terminal sequence of the activator e to the C-terminus of the activator g could increase the specific activity of L-NHase by178.40%.