Studies On Two Different Kinds Of Cyclic Amidases From Pseudomonas Putida YZ26

Cyclic amidohydrolase (cyclic amidase EC.3.5.2) is one of the most important hydrolase in organisms. Although the substrate specificity of these enzymes is quite different, the common character is to hydrolyze the cyclic amide bond in various organic acids outside of peptides or proteins. A report about the structural analysis of 16 cyclic amide amidohydrolases indicated that the rigidly conserved residues of these enzymes usually contained one aspartic acid and four histidines. A comparison of 3D conformation recently determined revealed that all of these enzymes have a structural core consisting of a (a/β)₈-barrel and a similar active site. They are involved in the metabolism of pyrimidine and purine in vivo and also in the bioconversion of amino acids or organic acid in vitro in industrial fields.According to the characteristics of optimal substrates, the superfamily of cyclic amidohydrolases may be divided into two subfamily, cyclic ureide hydrolase and cyclic imide hydrolase. The former includes hydantoinase, dihydropyrimidinase, allantoinase and dihydroorotase, the latter is only a cyclic imide hydrolase which is different from the former on the molecular mass of subunit, amino acid sequence and substrate specificity and so on. Based on our previous work, some results were abstracted as follows.1. The classification and identification of the enzyme-producing strainFrom the morphological observation, the strain reveals a single flagellum grown at the polar of cells, bacillus and Gram-negative. It was also indicated from physico-chemical properties that the oxidase and catalase reaction of the strain were positive and the glucose oxidation test was as a acid-producing type. The most important date is from 16S rDNA sequence analysis.Based on the homologous comparation from BLAST searching, the strain is named Pseudomonas putida YZ-26.2. Cloning and expression of D-hydantoinase gene as well as the purification and properties of the enzymeA 1440bp ORF encoding D-hydantoinase from Pseudomonas putida YZ-26 was amplified by PCR with the genomic DNA as the template. In view of Blast searching and homology comparison, the gene sequence of the D-hydantoinase is a new one and has been deposited in GenBank (Accession No: AY387829). The deduced amino acid sequence has an identity of 90.1% with the D-hydantoinase from Pseudomonas putdia CCRC 12857. Then, the gene was cloned into different expression vectors, such as pET3a, pET32M, pGEX-4T-l, pET28a and the recombinants were expressed in E.coli BL21. From the comparison of the activity of non-fusion and fusion D-hydantoinases, the N-terminal region seems to be more important than C-terminus. A rapid and efficient purification procedure for the non-fusion enzyme was performed by a three-step procedure: ammonium sulfate fractionation, followed by Phenyl Sepharose hydrophibic interaction chromatography and Sephacryl S-200 size exclusion chromatography. With the above processes, the overall recovery of enzymatic activity was 54.4 % and the specific activity for substrate DL-hydantoin achieved 16 U/mg with more than 95 % purity as estimated by SDS-PAGE analysis. On the contrary, the fusion enzyme was purified with Ni2+-NTA agarose affinity column and Sephacryl-S-200 size-exclusion chromatography. The overall recovery of the enzymatic activity reached 54.12 %, the specific activity for substrate DL-hydantoin was about 4.05 U/mg. The enzyme is a homologous dimer with a molecular mass 103 kDa as determined by both on native-PAGE and size-exclusion chromatography. The enzyme was stable at pH 6-12 with its optimum pH at 9.5. The optimum temperature of the enzyme was at 45°Cand it rapidly lost its activity over 55 °C. The effect of divalent metal ions on the enzyme activity displayed that Co2+, Mn2+ and Ni2+ was obviously as the activator, whilst Zn2+was a inhibitor. Moreover, the dimer enzyme can be partly dissociated at 1 mM Zn2+ with losing 30-40% activity, as determined by native-PAGE and size-exclusion chromatography.In addition, the D-hydantoinase gene from Pseudomonas putdia YZ-26 and the carbamoylase gene from Sinorhizobium morelense SS-ori were connected together to inserted into the vector pET28a and then the recombinant was expressed in E.coli to produce both HisTagged-Hydantoinase-Carbamoylase. The preliminary result revealed that when p-hydroxyl-phenylhydantoin as the substrate, the activity of the connected enzyme was slightly lower than that of the wild strain involving two separated enzymes described above.3. Cloning and expression of cyclic imide hydrolase gene as well as the purification and properties of the enzymeThe total chromosomal DNA isolated from Pseudomonas putdia YZ-26 was digested with EcoR I, and the fragments of 2 9kb were ligated into the corresponding sites of pUC118 as well as transformed into E.coli JM109. Transformants were initially screened by the white/blue color with X-gal. Subsequently, the imidase activity was detected by colorimetric method using DL-hydantoin as the substrate in 96-well microplate.The cyclic imide hydrolase(CIH) activity was found from a colony containing the inserted fragment of pUS804. The result pointed out from DNA sequence analysis that an open reading frame (ORF) of 879bp was displayed in the 2694bp fragment The ORF corresponds to 293 amino acid residues with a calculated molecular weight of 33.71 lkD. The gene sequence is a new one and has been deposited in GenBank (Accession No: DQ093858).The deduced amino acid sequence of CIH was blasted in GenBank and Swiss-Prot database. The comparison of sequence homology shows that there are 78% identity with the imidase from Alcaligenes eatrophus and has 80% identity with 20 amino acid at N-terminus of the imidase from Blastobacter sp.AXl p-4. The CIH gene was amplified by PCR and inserted into the vector pET32M. The engineered strain pEI/ï¿¡.co//BL21 can expressed the CIH which obviously has the activity with DL-hydantoin as the substrate. In addition, the His-tagged enzyme was also purified byfy Ione-step manipulation with Ni -NTA-agarose resin. The recovery of enzymatic activity reached 60.1 % with 38.5 U/mg protein, and 11.9-fold purification. The purified enzyme is a tetramer with a molecular mass of 141 kDa, as 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 the kinetic parameters of various substrates that the optimal substrate of the CIH is succinimide or maleimide, other than DL-hydantoin or dihydrouracil.Referring to the structure data of the cyclic amidase, 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 Histidines are essential for both the conformation and activity of the CIH. In addition, based on our previous work, the truncation and substitution of CIH at C-terminal region involving CIH292(-K), CIH291(-KK), CIH290(-RKK), CIH289 (-PRKK), KK292, 293EE, KK292, 293LL were performed as well. 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.