| Imidase (cyclic imide hydrolase,CIH,EC.3.5.2.16) is a member of cyclic amidase family which contains hydantoinase, dihydrouracil dehydrogenase, dihydroorotase, allantoinase and widely distributed in bacteria, fungi and molds. It's involved in ring-opening hydrolysis of cyclic imides to half-amides, and the resulting half-amide is hydrolyzed by half-amidase to dicarboxylates, which then undergoes further transformation through tricarboxylic acid (TCA) cyclic reaction.Imidase is attracting increasing attention as an important industrial enzyme to produce the unnatural amino acid, pyruvate and 3-carbamoyl-alpha-picolinic acid.Zinc is an important divalent metal iron in living organisms and an essential cofactor of many metabolic enzymes and transcription factors. Generally, zinc can be as agents to either stabilize protein conformation, or directly involved in catalytic activity of enzyme. According to the competition assay between PAR and imidase from Pseudomonas putida YZ-26, when PAR's concentration reached 1 mM, residual activity was at 16.6% in relation to the wild-type imidase and the binding constant for zinc/imidase complex was 9.5×108M-1.In order to explore the zinc-binding sites in imidase, we constructed a series of recombinant plasmids based on the Swiss-Model of imidase which are pMAL-s-cih247, pMAL-s-cih7.108,pMAL-s-cih7, pMAL-s-cih108,pMAL-s-cih86,pMAL-s-cih151 and then these recombinant plasmids were introduced into E.Coli BL21(DE3). These engineering bacteria can express the mutants of imidase as the soluble form in LB medium with 0.25 mM IPTG induction for 4～5 hours at 37℃. By the enzyme acitivity assay, H247/A, CC7,108/GG, C7/G, H86/A have been showed a little activities and C108/G retains 72% of the activity, while the H151/G contains the enzymatic activity by about 120%. The mutants of imidase were purified to homogeneousness by the following procedures:Amylose affinity column, Human Rhinocirus 3C Protease digestion, Superose-12 gel-filtration chromarography.Through the PAR competition assay, we compare the zinc-binding abilities of these mutants. The results have been shown that the zinc competition of H247/A, H86/A and CC7,108/GG were all markedly decreased, while mutant H151/G ability of for zinc to compete with PAR was slightly higher than wild-type imidase. In view of the enzyme activities, His247, His86 and Cys7,108 may be closely related to the zinc binding and catalytic activity of imidase. Subsequently, the results from that of circular dichroism of mutants and crystal structure of wild-type imidase have been corfirmed that His86, His90 and Asp 14 are involved in zinc binding and His247, together with Tyr23, is the key residues for substrate binding.Cysteines are commonly regarded as the most prevalent activity centers or the zinc-coordinating residues in most enzymes and play important roles in the organization and maintenance of protein tertiary structure by forming the disufide bond. Both two cysteine residues of imidase were modified by the DTNB and analytized by reduction and non-reduction SDS-PAGE. The results have been shown that these are two free cysteine residues in wild-type imidase. It is indicated that C108/Q as well as imidase, is a homologous tetramer and rentains the most of catalytic activity, while C7/G is composed of the monomer and polymer and CC7,108/GG is total of the polymer without any activity using the size exclusion column and zinc competition assay. Cys7 may more important than Cys108 in maintaining the state of imidase.
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