Bhat M A, Ishida T, Horiike K, Vaidyanathan C S, Nozaki M
Department of Biochemistry, Shiga University of Medical Science, Japan.
Arch Biochem Biophys. 1993 Feb 1;300(2):738-46. doi: 10.1006/abbi.1993.1102.
An enzyme which cleaves the benzene ring of 3,5-dichlorocatechol has been purified to homogeneity from Pseudomonas cepacia CSV90, grown with 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole carbon source. The enzyme was a nonheme ferric dioxygenase and catalyzed the intradiol cleavage of all the examined catechol derivatives, 3,5-dichlorocatechol having the highest specificity constant of 7.3 microM-1s-1 in an air-saturated buffer. No extradiol-cleaving activity was observed. Thus, the enzyme was designated as 3,5-dichlorocatechol 1,2-dioxygenase. The molecular weight of the native enzyme was ascertained to be 56,000 by light scattering method, while the M(r) value of the enzyme denatured with 6 M guanidine-HCl or sodium dodecyl sulfate was 29,000 or 31,600, respectively, suggesting that the enzyme was a homodimer. The iron content was estimated to be 0.89 mol per mole of enzyme. The enzyme was deep red and exhibited a broad absorption spectrum with a maximum at around 425 nm, which was bleached by sodium dithionite, and shifted to 515 nm upon anaerobic 3,5-dichlorocatechol binding. The catalytic constant and the Km values for 3,5-dichlorocatechol and oxygen were 34.7 s-1 and 4.4 and 652 microM, respectively, at pH 8 and 25 degrees C. Some heavy metal ions, chelating agents and sulfhydryl reagents inhibited the activity. The NH2-terminal sequence was determined up to 44 amino acid residues and compared with those of the other catechol dioxygenases previously reported.
一种能裂解3,5-二氯邻苯二酚苯环的酶已从洋葱伯克霍尔德菌CSV90中纯化至同质，该菌以2,4-二氯苯氧乙酸（2,4-D）作为唯一碳源生长。该酶是一种非血红素铁双加氧酶，催化所有检测的邻苯二酚衍生物的二醇裂解，在空气饱和缓冲液中，3,5-二氯邻苯二酚的特异性常数最高，为7.3 μM-1s-1。未观察到间位裂解活性。因此，该酶被命名为3,5-二氯邻苯二酚1,2-双加氧酶。通过光散射法确定天然酶的分子量为56,000，而用6 M盐酸胍或十二烷基硫酸钠变性的酶的M(r)值分别为29,000或31,600，表明该酶是同型二聚体。估计每摩尔酶的铁含量为0.89摩尔。该酶呈深红色，表现出宽吸收光谱，最大吸收峰在425 nm左右，用连二亚硫酸钠可使其褪色，在厌氧条件下结合3,5-二氯邻苯二酚后吸收峰移至515 nm。在pH 8和25℃下，3,5-二氯邻苯二酚和氧气的催化常数和Km值分别为34.7 s-1、4.4和652 μM。一些重金属离子、螯合剂和巯基试剂会抑制该活性。测定了该酶的氨基末端序列，直至44个氨基酸残基，并与先前报道的其他邻苯二酚双加氧酶的序列进行了比较。
