Primary sequence and activity analyses of a catalase from Ascaris suum

• Published in: Molecular and biochemical parasitology Vol. 95; no. 2; pp. 203 - 214
• Main Authors: Eckelt, Volker H.O., Liebau, Eva, Walter, Rolf D., Henkle-Dührsen, Kimberly
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15-09-1998
• Subjects: Amino Acid Sequence; amino acid sequences; animal parasitic nematodes; Animals; Ascaris suum; Ascaris suum - enzymology; Ascaris suum - genetics; Base Sequence; Catalase; Catalase - antagonists & inhibitors; Catalase - chemistry; Catalase - genetics; Catalase - metabolism; Cattle; complementary DNA; Detoxification; DNA, Complementary; enzyme activity; enzyme inhibitors; Enzyme Inhibitors - pharmacology; genbank/y10611; Hydrogen peroxide; Hydrogen Peroxide - metabolism; Hydrogen-Ion Concentration; metabolic detoxification; Molecular Sequence Data; Molecular Weight; NADP - metabolism; nucleotide sequences; Oxidative stress; Peroxidase - metabolism; physicochemical properties; recombinant proteins; Recombinant Proteins - biosynthesis; Recombinant Proteins - metabolism; Sequence Alignment; Spectrometry, Fluorescence; Spectrophotometry; stress; Oxidative stress; Hydrogen peroxide; Catalase; AsCAT, catalase from Ascaris suum; rAsCAT, recombinant AsCAT; CAT, catalase; Detoxification; Ascaris suum; PCR, polymerase chain reaction; ROS, reactive oxygen species; BLC, beef liver catalase; tBOOH, t-butyl hydroperoxide
• ISSN: 0166-6851; 1872-9428
• DOI: 10.1016/S0166-6851(98)00092-9

A complete cDNA encoding the catalase (EC 1.11.1.6) has been isolated from the parasitic nematode
Ascaris suum (AsCAT). The active-site residues, the residues involved in ligand interaction, and NADPH-binding residues of the bovine liver catalase-type enzyme are highly conserved in the AsCAT predicted amino acid sequence. To confirm that the AsCAT cDNA encodes a functional enzyme, active recombinant protein (rAsCAT) was produced in a procaryotic expression system. The subunit molecular mass of the purified recombinant protein (rAsCAT) was determined to be ∼60 kDa. According to gel filtration, the molecular mass of the active enzyme is 240 kDa, indicating that the catalase subunits form a homotetramer in solution. The optical spectrum of rAsCAT shows a typical ferric haem spectrum with a Soret band at 407 nm. Fluorescence spectroscopy demonstrates that rAsCAT binds NADPH. rAsCAT has catalase activity with hydrogen peroxide over a broad pH range, with a specific activity of 37 800 U mg
−1. In addition to its catalase activity, rAsCAT displays peroxidase activity using the substrates
t-butyl hydroperoxide and
o-dianisidine. The haem ligands NaN
3 and KCN caused a 50% inhibition of catalase activity at 9 and 19
μM, respectively. In the presence of a H
2O
2-generating system, catalase activity of rAsCAT was inhibited by 3-aminotriazole, phenolic compounds, and drugs.