Gene Cloning, Fusion Expression And Biochemical Properties Of A Cyclic Imide Hydrolase

Cyclic imide hydrolase (CIH, EC.3.5.2.16) is a member of cyclic amidase family and its subunit molecular mass (Mr) is about 35 kDa, which is usually less than that of common cyclic amidases (50-60 kDa), i.e., dihydropyrimidinase, hydantoinase etc. Based on the known data, the CIH is obviously different from bacterial D-hydantoinases in active site, amino acid sequence, substrate specificity, subunit Mr and so on. It was reported that the enzyme from Blasterobacter sp. A17p-4 participated in cyclic imide metabolism, involving in the ring-opening hydrolysis of cyclic imides to half-amide and successively half-amide amidohydrolysis to dicarboxylate and finally to the tricarboxylic acid cycle. The CIH displays the potential application not only in the synthesis of intermediates of drugs or in the detoxication of xenobiotics in vivo, but also in the transformation of organic acids or chiral compounds.An open reading frame (ORF) encoding the CIH was successfully cloned from Pseudomonas putdia YZ-26, which was screened by this Laboratory. It is indicated form DNA sequence analysis that the CIH ORF contains 879 bp corresponding 293 amino acid residues with a calculated molecular weight of 33.7 kDa. In view of Blast searching and homology comparison, the gene sequence of CIH is a new one and has beendeposited in GenBank (Accession No: DQ093858). The deduced amino acid sequence has an identity of 78 % with the CIH from Alcaligenes eutrophus II2R4 and 80 % with N-terminal 20 amino acids of CIH from Blastobacter sp A17p-4.The ORF of CIH was introduced into vector pET-32M to generate a recombinant plasmid, pEI. Then the engineered strain pEI lE.coli BL21(DE3) was incubated and induced in LB medium at 37 °C for 5 h by adding the final concentration of 0.25 mmol/L IPTG. The results show that the target product is soluble and active with the yield of about 10 %, compared with the total proteins in cells and the enzymatic activity reaches 5.19 U/ mL with Hydantoin as the substrate. The enzyme was purified with Ni2+—NTA agarose affinity column and Sephacryl —S~ 200 size-exclusion chromatography. With above procedures, the overall recovery of enzymatic activity reached 60.1 %, the specific activity was about 38.5 U/mg protein, and the fold of purification was 11.9. The purified enzyme is a tetramer with Mr of 141 kDa determined by size-exclusion chromatography. The optimal pH of the CIH is at 9.0 and the optimal temperature is at 50 °C. It is shown from the kinetic parameters of various substrates that the optimal substrate of the CIH is Succinimide or Maleimide, other than Hydantoin or Dihydrouracil.Referring to the structure data of cyclic amidases reported, histidine mutation was conducted as follows: H86A, H90A, H247A, H266A,H270A. Unfortunately, all of mutant enzymes have no detectable activity in the comparison of the wild-type CIH, possibly indicating that these Hiss are essential for both the conformation and activity of the CIH. In addition, based on our previous work on D-hydantoinase, the truncation and substitution of CIH (293 residues) at C-terminal region were tested to explore their importance for the conformation or activity. They were CIH292(-K), CIH291(-KK), CIH290(-RKK) > CIH289(-PRKK), KK292,293EE. KK292,293LL and their activities are compared with the intact CIH respectively. Meanwhile, it is indicated from CD and fluorescent spectra that the conformational change of these mutant enzymes is basically accordant with their resident activity, suggesting that the positive charges at C-terminus are important for both the conformation and activity.