Rocha E R, Smith C J
Department of Microbiology and Immunology, School of Medicine, East Carolina State University, Greenville, North Carolina 27858-4354, USA.
J Bacteriol. 1995 Jun;177(11):3111-9. doi: 10.1128/jb.177.11.3111-3119.1995.
A single catalase enzyme was produced by the anaerobic bacterium Bacteroides fragilis when cultures at late log phase were shifted to aerobic conditions. In anaerobic conditions, catalase activity was detected in stationary-phase cultures, indicating that not only oxygen exposure but also starvation may affect the production of this antioxidant enzyme. The purified enzyme showed a peroxidatic activity when pyrogallol was used as an electron donor. It is a hemoprotein containing one heme molecule per holomer and has an estimated molecular weight of 124,000 to 130,000. The catalase gene was cloned by screening a B. fragilis library for complementation of catalase activity in an Escherichia coli catalase mutant (katE katG) strain. The cloned gene, designated katB, encoded a catalase enzyme with electrophoretic mobility identical to that of the purified protein from the B. fragilis parental strain. The nucleotide sequence of katB revealed a 1,461-bp open reading frame for a protein with 486 amino acids and a predicted molecular weight of 55,905. This result was very close to the 60,000 Da determined by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified catalase and indicates that the native enzyme is composed of two identical subunits. The N-terminal amino acid sequence of the purified catalase obtained by Edman degradation confirmed that it is a product of katB. The amino acid sequence of KatB showed high similarity to Haemophilus influenzae HktE (71.6% identity, 66% nucleotide identity), as well as to gram-positive bacterial and mammalian catalases. No similarities to bacterial catalase-peroxidase-type enzymes were found. The active-site residues, proximal and distal hemebinding ligands, and NADPH-binding residues of the bovine liver catalase-type enzyme were highly conserved in B. fragilis KatB.
当处于对数生长期后期的脆弱拟杆菌培养物转移到需氧条件下时，该厌氧菌会产生单一的过氧化氢酶。在厌氧条件下，稳定期培养物中检测到了过氧化氢酶活性，这表明不仅氧气暴露，饥饿也可能影响这种抗氧化酶的产生。当以连苯三酚作为电子供体时，纯化后的酶表现出过氧化物酶活性。它是一种血红蛋白，每个全酶分子含有一个血红素分子，估计分子量为124,000至130,000。通过筛选脆弱拟杆菌文库以互补大肠杆菌过氧化氢酶突变体（katE katG）菌株中的过氧化氢酶活性，克隆了过氧化氢酶基因。克隆的基因命名为katB，编码的过氧化氢酶的电泳迁移率与脆弱拟杆菌亲本菌株纯化蛋白的电泳迁移率相同。katB的核苷酸序列显示有一个1461 bp的开放阅读框，编码一个含486个氨基酸、预测分子量为55,905的蛋白质。这一结果与通过纯化过氧化氢酶的十二烷基硫酸钠 - 聚丙烯酰胺凝胶变性电泳测定的60,000 Da非常接近，表明天然酶由两个相同的亚基组成。通过埃德曼降解获得的纯化过氧化氢酶的N端氨基酸序列证实它是katB的产物。KatB的氨基酸序列与流感嗜血杆菌HktE高度相似（同一性为71.6%，核苷酸同一性为66%），也与革兰氏阳性菌和哺乳动物的过氧化氢酶相似。未发现与细菌过氧化氢酶 - 过氧化物酶型酶有相似性。牛肝过氧化氢酶型酶的活性位点残基、近端和远端血红素结合配体以及NADPH结合残基在脆弱拟杆菌KatB中高度保守。
