de Souza M L, Sadowsky M J, Wackett L P
Department of Biochemistry, University of Minnesota, St. Paul 55108, USA.
J Bacteriol. 1996 Aug;178(16):4894-900. doi: 10.1128/jb.178.16.4894-4900.1996.
Pseudomonas sp. strain ADP metabolizes atrazine to carbon dioxide and ammonia via the intermediate hydroxyatrazine. The genetic potential to produce hydroxyatrazine was previously attributed to a 1.9-kb AvaI DNA fragment from strain ADP (M. L. de Souza, L. P. Wackett, K. L. Boundy-Mills, R. T. Mandelbaum, and M. J. Sadowsky, Appl. Environ. Microbiol. 61:3373-3378, 1995). In this study, sequence analysis of the 1.9-kb AvaI fragment indicated that a single open reading frame, atzA, encoded an activity transforming atrazine to hydroxyatrazine. The open reading frame for the chlorohydrolase was determined by sequencing to be 1,419 nucleotides and encodes a 473-amino-acid protein with a predicted subunit molecular weight of 52,421. The deduced amino acid sequence matched the first 10 amino acids determined by protein microsequencing. The protein AtzA was purified to homogeneity by ammonium sulfate precipitation and anion-exchange chromatography. The subunit and holoenzyme molecular weights were 60,000 and 245,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography, respectively. The purified enzyme in H2(18)O yielded [18O]hydroxyatrazine, indicating that AtzA is a chlorohydrolase and not an oxygenase. The most related protein sequence in GenBank was that of TrzA, 41% identity, from Rhodococcus corallinus NRRL B-15444R. TrzA catalyzes the deamination of melamine and the dechlorination of deethylatrazine and desisopropylatrazine but is not active with atrazine. AtzA catalyzes the dechlorination of atrazine, simazine, and desethylatrazine but is not active with melamine, terbutylazine, or desethyldesisopropylatrazine. Our results indicate that AtzA is a novel atrazine-dechlorinating enzyme with fairly restricted substrate specificity and contributes to the microbial hydrolysis of atrazine to hydroxyatrazine in soils and groundwater.
假单胞菌属ADP菌株通过中间产物羟基阿特拉津将阿特拉津代谢为二氧化碳和氨。此前认为产生羟基阿特拉津的遗传潜力归因于来自ADP菌株的一个1.9 kb的AvaI DNA片段（M. L. de Souza、L. P. Wackett、K. L. Boundy-Mills、R. T. Mandelbaum和M. J. Sadowsky，《应用与环境微生物学》61:3373 - 3378，1995年）。在本研究中，对该1.9 kb AvaI片段的序列分析表明，一个单一的开放阅读框atzA编码一种将阿特拉津转化为羟基阿特拉津的活性。通过测序确定氯水解酶的开放阅读框为1419个核苷酸，编码一个473个氨基酸的蛋白质，预测亚基分子量为52421。推导的氨基酸序列与通过蛋白质微量测序确定的前10个氨基酸相匹配。蛋白质AtzA通过硫酸铵沉淀和阴离子交换色谱法纯化至同质。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳和凝胶过滤色谱法分别测定亚基和全酶的分子量为60000和245000。在H2(18)O中的纯化酶产生了[18O]羟基阿特拉津，表明AtzA是一种氯水解酶而非加氧酶。GenBank中最相关的蛋白质序列是来自珊瑚红球菌NRRL B - 15444R的TrzA，同一性为41%。TrzA催化三聚氰胺的脱氨以及去乙基阿特拉津和去异丙基阿特拉津的脱氯，但对阿特拉津无活性。AtzA催化阿特拉津、西玛津和去乙基阿特拉津的脱氯，但对三聚氰胺、特丁津或去乙基去异丙基阿特拉津无活性。我们的结果表明AtzA是一种新型的阿特拉津脱氯酶，底物特异性相当有限，有助于土壤和地下水中阿特拉津微生物水解为羟基阿特拉津。
