Study On Properties Of Recombinant Hexahistidine-tagged 5-Aminolevulinate Dehydratase From Bacillus Subtilis

All tetrapyrroles such as hemes, chlorophylls and vitamin B12 play the important role in the life. The first common intermediate for all tetrapyrroles is 5-aminolevulinate (ALA). Two molecules of ALA is condensed to one molecule porphobilinogen (PBG), catalyzed by 5-aminolevulinic acid dehydratase (ALAD), and also known as porphobilinogen synthase.ALAD enzymes were purified and characterized from several organisms. It is a metal-protein. Different metal ions were used in ALAD from the different organisms. ALAD is octamers in microbes and mammalians, and hexamers in plant. The genes encoding ALAD were cloned from about a hundred of organisms, which showed about 30% homogeneity. The essential amino acid residues for catalysis were determined by site-directed mutation using the recombinant system. The crystal structure of several ALADs revealed the high similarity of the catalytic cleft. The hemB from Bacillus subtilus encoding ALAD was cloned. The amino acid alignment displays that ALAD from B. subtilis is high similarity to that from Escherichia coli. However, the active site of the enzyme is a little different from that from E. coli. The properties of ALAD from B. subtilus were not known. Here, we reported the purification and characterization of recombinant B. subtilus ALAD overexpressed in E. coli. The rapid purification and characterization of this enzyme will facilitate to biosynthesize porphobilinogen easily in vitro. As a substrate, porphobilinogen is used in the couple assay of the following enzyme, and also applied in the biosynthesis of the downstream of intermediates, which is applied as the medicine in the cancer therapy. The all results in this study are displayed as followed:1. The construction of the expression vector encoding ALAD from B. subtilisThe hemB from B. sublitis was amplified by PCR and was completely identical to the reported hemB gene by sequencing. The purified hemB with Nde I and EcoR I digestion was inserted into pET-28a plasmid with the same manner to yield the recombinant vector, named pET28a-ALAD which encodes ALAD with His6-tag at N-terminus.2. The overexpression and one-step purification of Bacillus subtilus ALADThe constructed expression vector was transformed into Escherchia coli BL21 (DE3). Upon the induction with 0.5 mM IPTG for 5h, recombinant ALAD was expressed at high level.A specific band at 41kD was displayed on SDS-PAGE.About 164U·mg-1 specific activity for ALAD was detected from the crude of the induced strains, suggesting that the His6-tagged ALAD was overexpressed in active form, and His6-tag fusion did not affect the enzyme activity obviously. Using the His6-tag at N-terminus, ALAD was purified by one-step purification, the affinity chromatography Ni-NTA. The high purific ALAD was obtained, as shown by SDS-PAGE.3. Characterization of ALAD from B. subtilisThe subunit molecular weight of Bacillus subtilus ALAD was about 41kDa, as estamited from SDS-PAGE analysis, and the holo-enzyme molecular mass of the enzyme was about 330 kDa, as calculated by PAGE. The results indicated the protein consists of eight identical subunits. Its optimum pH was about 8.0. Km and Vmax for ALAD was expected about 7.4mM and 0.54mM·h-1 respectively. The purified enzyme retained 80% of enzyme activity at 55℃for 10min. The metal ions including K+,Zn2+,Mg2+,Li+,Fe3+ and Mn2+ enhanced the activity obviously, whereas Cu2+ and Hg2+ caused the complete loss of the activity. The enzyme activity was increased with the concentration ofβ-mercaptoethanol up to 10mmol/L, possibly due to the prohibition of oxidation for porphobilinogen. However, the ALAD was entirely inactive in the presence of reducing agent dithiothreitol at high concentration up to 100 mmol/L, deducing that the high concentration of dithiothreitol affected the binding metal ion with Cys residue. Decoration of the ALAD with several amino-acid specific reagents demonstrated that Lys and Cys residues were essential for catalysis, since the activity of ALAD treated with His and Lys decorating reagents were not detected. His and Ser residues were not critical for enzyme catalysis, resulting from the assay that the activity of the ALAD retained about 38% and 36% respectively, suggesting the decorating reagents may cause the protein conformational change.