Characterization of thermostable tyrosine phenol-lyase from an obligatory symbiotic thermothile, Symbiobacterium sp. SC-1

Abstract

Tyrosine phenol-lyase of thermophilic Symbiobacterium sp. SC-1, which is obligately and symbiotically dependent on thermophilic Bacillus sp. SK-1, was purified and characterized. The enzyme is composed of four identical subunits and contains approximately 1 mol of pyridoxal 5′-phosphate (PLP) per mol subunit as a cofactor. The enzyme showed absorption maxima at 330 and 420 nm, and lost this absorption profile by treatment with phenylhydrazine. The apparent dissociation constsnt, K′D, for PLP was determined with the apoenzyme to be about 1.2 μM. The isoelectric point was 4.9. The optimal temperature and pH for the α,β-elimination of L-tyrosine were found to be 80°C and pH 8.0, respectively. The substrate specificity of the enzyme was very broad: L-amino acids including L-tyrosine, 3,4-dihydroxyphenyl-L-alanine (L-DOPA), L-cysteine, L-serine, S-methyl-L-cysteine, β-chloro-L-alanine, and S-(o-nitrophenyl)-L-cysteine all served as substrates. D-Tyrosine and D-serine were also decomposed into pyruvic acid and ammonia at rates of 7% and 31% relative to their corresponding L-enantiomers, respectively. D-Alanine, which was inert as a substrate in α, β-elimination, was the only D-amino acid racemized by the enzyme. The Km values for L-tyrosine, L-DOPA, S-(o-nitrophenyl)-L-cysteine, β-chloro-L-alanine, and S-methyl-L-cysteine were 0.19, 9.9, 0.36, 12, and 5.5 mM, respectively.