Expression, purification and characterization of arginase from Helicobacter pylori in its apo form.
Arginase, a manganese-dependent enzyme that widely distributed in almost all creatures, is a urea cycle enzyme that catalyzes the hydrolysis of L-arginine to generate L-ornithine and urea. Compared with the well-studied arginases from animals and yeast, only a few eubacterial arginases have been characterized, such as those from H. pylori and B. anthracis. However, these enzymes used for arginase activity assay were all expressed with LB medium, as low concentration of Mn(2+) was detectable in the medium, protein obtained were partially Mn(2+) bonded, which may affect the results of arginase activity assay. In the present study, H. pylori arginase (RocF) was expressed in a Mn(2+) and Co(2+) free minimal medium, the resulting protein was purified through affinity and gel filtration chromatography and the apo-form of RocF was confirmed by flame photometry analysis. Gel filtration indicates that the enzyme exists as monomer in solution, which was unique as compared with homologous enzymes. Arginase activity assay revealed that apo-RocF had an acidic pH optimum of 6.4 and exhibited metal preference of Co(2+)>Ni(2+)>Mn(2+). We also confirmed that heat-activation and reducing regents have significant impact on arginase activity of RocF, and inhibits S-(2-boronoethyl)-L-Cysteine (BEC) and Nω-hydroxy-nor-Arginine (nor-NOHA) inhibit the activity of RocF in a dose-dependent manner.
Department of Clinical Microbiology and Immunology, College of Medical Laboratory, Third Military Medical University, Chongqing, China.
Jinyong
J
Zhang
Xiaoli
X
Zhang
Chao
C
Wu
Dongshui
D
Lu
Gang
G
Guo
Xuhu
X
Mao
Ying
Y
Zhang
Da-Cheng
DC
Wang
Defeng
D
Li
Quanming
Q
Zou
D000595:Amino Acid Sequence; D001051:Apoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001119:Arginase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001897:Boronic Acids / Q000494:pharmacology; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D016480:Helicobacter pylori / Q000201:enzymology* / Q000235:genetics*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D055503:Protein Multimerization / Q000187:drug effects; D020836:Protein Structure, Quaternary; D019163:Reducing Agents / Q000494:pharmacology
C425062:(2-boronoethyl)-cysteine; D001051:Apoenzymes; D001426:Bacterial Proteins; D001897:Boronic Acids; D004791:Enzyme Inhibitors; D008670:Metals; C404735:N(omega)-hydroxynorarginine; D019163:Reducing Agents; D001120:Arginine; C402431:RocF protein, Helicobacter pylori; D001119:Arginase