Characterization of ergothionase from Burkholderia sp. HME13 and its application to enzymatic quantification of ergothioneine.
We identified ergothionase, which catalyzes conversion of ergothioneine to thiolurocanic acid and trimethylamine, in a newly isolated ergothioneine-utilizing strain, Burkholderia sp. HME13. The enzyme was purified and its N-terminal amino acid sequence was determined. Based on the amino acid sequence, the gene encoding the enzyme was cloned and expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity and characterized. The enzyme consisted of four identical 55-kDa subunits. The enzyme showed maximum activity at pH 8.0 and 65 °C and was stable between pH 7.0 and pH 10.0 and up to 60 °C. The enzyme acted on ergothioneine (K m: 19 μM, V max: 270 μmol/min/mg), but not D-histidine, L-histidine, D-tyrosine, L-tyrosine, D-phenylalanine, or L-phenylalanine. The enzyme was activated by BaCl2 and strongly inhibited by CuSO4, ZnSO4, and HgCl2. The amino acid sequence of ergothionase showed 23 % similarity to histidine ammonia-lyase (HAL) from Pseudomonas putida and 17 % similarity to phenylalanine ammonia-lyase (PAL) from parsley. However, the tripeptide sequence, Ala-Ser-Gly, which is important for catalysis in both HAL and PAL, was not conserved in ergothionase. The application of ergothionase for the quantification of ergothioneine contained in practical food and blood samples was investigated by performing a recovery test. Satisfactory recovery data (98.7-104 %) were obtained when ergothioneine was added to extract of tamogitake and hemolysis blood.
Multidisciplinary Science Cluster, Research and Education Faculty, Kochi University, B200 Monobe, Nankoku, Kochi 783-8502, Japan. hmura@kochi-u.ac.jp
Hisashi
H
Muramatsu
Hidenori
H
Matsuo
Naoki
N
Okada
Momoko
M
Ueda
Hiroaki
H
Yamamoto
Shin-ichiro
S
Kato
Shinji
S
Nagata
D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D020536:Enzyme Activators / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D004880:Ergothioneine / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008190:Lyases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D021122:Protein Subunits; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D004269:DNA, Bacterial; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D021122:Protein Subunits; D011994:Recombinant Proteins; D004880:Ergothioneine; D008190:Lyases