Identification Of A Catalase-Phenol Oxidase In Red Amaranth (Amaranthus Cruentus), A Betalain-Producing Plant

Betalains are a group of nitrogen-containing pigments containing red-violet betacyanins and yellow betaxanthins. Betalains replace anthicyanins and color plants in most families of Caryophyllales. Betalains are used in food and pharmaceutical industries because of their antioxidant and radical scavenging properties. Their biosynthetic pathway has generally been proposed to begin with the hydroxylation of L-tyrosine through monophenolase activity of PPO-type tyrosinase, but the dispute remains over the enzyme(s) for this first step. We attempt to purify the enzymes hydroxylating L-tyrosine and clone their genes to elucidate the first step of betalain biosynthesis.An enzyme with tyrosinase activity was purified to apparent homogeneity through gel slice from leaves of Amaranthus cruentus, a betalain plant. The purified enzyme appeared to be a homotrimeric protein composed of subunits of about 58 kDa estimated by SDS-PAGE and native PAGE. The purified enzyme demonstrated not only the catalase activity toward H2O2, but also the monophenolase activity toward L-tyrosine and diphenolase activity toward L-DOPA in vitro. The three activities of this enzyme had different optimal temperatures, pH and Km values. Its catalase and phenol oxidase activities were inhibited by common classic catalase and tyrosinase inhibitors except tropolone, respectively.The Nano-LC-MS/MS analysis of the purified enzyme gave five clear peptides. A MASCOT database search targeted surprisingly all five peptides to catalases, including those from two betalain-producing plants Suaeda salsa and Mesembryanthemum crystallinum.Based on the mRNA sequences of two betalain-producing plant catalases, we cloned a complete cDNA sequence (1951 bp) using RT-PCR and RACE, and accordingly cloned an entire CDS. The RACE CDS and entire CDS encoded deductively a peptide of 492 amino acids, identically. As expected, the peptide contained not only all five peptide fragments determined by Nano-LC-MS/MS, but also both catalase active site and possible phenol oxidase active site. Thus, the purified enzyme was named as AcCATPO and its gene, AcCATPO.In order to know whether this new group of catalases exists in non-betalain plants, we compared the amino acid sequences of our AcCATPO and CATPOs known with those of plant catalases collected from GenBank. The results revealed that the potential oxidase active site, besides catalase active site, was even present in the catalases of non-betalain plants. This suggests that the 4th group of catalases, CATPOs, is present in plant in general.To test whether AcCATPO is involved in the L-tyrosine hydroxylation, the first step of betalain biosynthetic pathway, we examined the correlation between AcCATPO mRNA expression level and betalain (betacyanin and betaxanthin) contents in the leaf green sectors and red sectors of common amaranth (A. tricolor). We observed a positive correlation between AcCATPO transcript abundance and the ratio of betaxanthins to betacyanins, suggesting that AcCATPO might be mainly involved in the biosynthesis of betaxanthins.These data shows that the fourth group catalase, catalase-phenol oxidase, is also present in plant, and might be involved in the first step of betalain biosynthesis in betalain-forming plants. Successful identification of the fourth group catalase in plant opens a window to investigate the function of this new class of plant catalases, and in particular its role in betalain biosynthesis in betalain-producing plants.