Sanz Rosario, Marı N Irma, Ruiz-Santa-Quiteria Jose A, Orden Jose A, Cid Dolores, Diez Rosa M, Silhadi K Souad, Amils Ricardo, de la Fuente Ricardo
Departamento Patologı́a Animal I, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain1.
Centro de Biologı́a Molecular, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain2.
Microbiology (Reading). 2000 Feb;146 ( Pt 2):465-475. doi: 10.1099/00221287-146-2-465.
Degenerate oligonucleotide primers based on internal peptide sequences obtained by HPLC from purified Staphylococcus aureus catalase were used to locate the S. aureus and S. aureus subsp. anaerobius kat regions by PCR. Southern hybridization analysis with a probe derived from a 1.1 kb PCR-amplified fragment showed that a single copy of the putative catalase gene was present in the S. aureus and S. aureus subsp. anaerobius chromosome. The nucleotide sequence of S. aureus katA revealed a 1518 bp open reading frame for a protein with 505 amino acids and a predicted molecular mass of 58347 Da, whereas S. aureus subsp. anaerobius katB is 1368 nt long and encodes a polypeptide of 455 amino acids with a predicted molecular mass of 52 584 Da. These catalases are highly homologous to typical monofunctional catalases from prokaryotes. The active-site residues, proximal and distal haem-binding ligands and NADPH-binding residues of the bovine liver catalase-type enzyme were highly conserved in S. aureus KatA. Escherichia coli cells carrying cloned katA had a catalase activity approximately 1000 times that of untransformed E. coli, but no detectable increase in catalase activity was observed with E. coli carrying cloned katB. Northern blotting showed the presence of a kat-specific transcript in S. aureus subsp. anaerobius, suggesting that the lack of catalase activity in this bacterium is due to a post-transcriptional alteration. Compared to the nucleotide sequence of katA, katB showed a single base-pair deletion and six mis-sense mutations, and these alterations were present in three other S. aureus subsp. anaerobius strains analysed. The deletion, located at 1338 bp from the initiation codon, originates a shift of the nucleotide reading frame and is responsible for the premature translation termination at 1368 bp, generating a KatB polypeptide 50 amino acid residues shorter than KatA. Moreover, four of the mis-sense mutations present in katB lead to non-conservative amino acid replacements, the most significant being that located at residue 317 (Pro in KatA-->Ser in KatB) because the affected amino acid is involved in determining the proximal haem-binding site. Both the main alterations found in KatB (the deletion and the substitution in residue 317) seem to contribute to the lack of catalase activity in S. aureus subsp. anaerobius, as deduced from results obtained with chimeric catalase constructs.
基于通过高效液相色谱法从纯化的金黄色葡萄球菌过氧化氢酶中获得的内部肽序列设计的简并寡核苷酸引物，用于通过聚合酶链反应（PCR）定位金黄色葡萄球菌和金黄色葡萄球菌厌氧亚种的kat区域。用源自1.1 kb PCR扩增片段的探针进行Southern杂交分析表明，推定的过氧化氢酶基因的单拷贝存在于金黄色葡萄球菌和金黄色葡萄球菌厌氧亚种的染色体中。金黄色葡萄球菌katA的核苷酸序列揭示了一个1518 bp的开放阅读框，编码一个含有505个氨基酸、预测分子量为58347 Da的蛋白质，而金黄色葡萄球菌厌氧亚种的katB长1368 nt，编码一个含有455个氨基酸、预测分子量为52584 Da的多肽。这些过氧化氢酶与原核生物中的典型单功能过氧化氢酶高度同源。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结合残基在金黄色葡萄球菌KatA中高度保守。携带克隆的katA的大肠杆菌细胞的过氧化氢酶活性比未转化的大肠杆菌高约1000倍，但携带克隆的katB的大肠杆菌未观察到过氧化氢酶活性有可检测到的增加。Northern印迹显示金黄色葡萄球菌厌氧亚种中存在kat特异性转录本，表明该细菌中缺乏过氧化氢酶活性是由于转录后改变。与katA的核苷酸序列相比，katB显示一个碱基对缺失和六个错义突变，并且这些改变存在于分析的其他三个金黄色葡萄球菌厌氧亚种菌株中。该缺失位于起始密码子下游1338 bp处，导致核苷酸阅读框移位，并导致在1368 bp处提前终止翻译，产生一个比KatA短50个氨基酸残基的KatB多肽。此外，katB中存在的四个错义突变导致非保守氨基酸替换，最显著的是位于残基317处的突变（KatA中的Pro→KatB中的Ser），因为受影响的氨基酸参与确定近端血红素结合位点。从嵌合过氧化氢酶构建体获得的结果推断，在KatB中发现的主要改变（缺失和残基317处的替换）似乎都导致金黄色葡萄球菌厌氧亚种中缺乏过氧化氢酶活性。
