Exploring the structure and function of the mycobacterial KatG protein using trans-dominant mutants
Exploring the structure and function of the mycobacterial KatG protein using trans-dominant mutants
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DOI：
10.1128/aac.47.1.188-195.2003
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发表时间：
2003-01-01
影响因子：
4.9
通讯作者：
Morris, S
中科院分区：
文献类型：
--
作者：
DeVito, JA;Morris, S
为了探索分枝杆菌过氧化氢酶-过氧化物酶(KatG)的结构和功能，我们采用了katG分体二倍体显性-负性分析的遗传方法。用不同的katG点突变体(从低拷贝数质粒表达)转化牛分枝杆菌BCG后，细胞提取物中的过氧化物酶和过氧化氢酶活性降低。这些酶活性的降低通常与抗结核药物异烟肼(INH)耐药性的增加有关。然而，对于N138S反式显性突变体，过氧化氢酶-过氧化物酶活性显著降低，同时保持了对异烟肼的敏感性。反式显性需要从多拷贝质粒中表达katG，而野生型BCG染色体上其他地方整合的katG突变体不能证明这一点。通过质粒交换逆转突变表型，表明过氧化氢酶-过氧化物酶缺乏症发生在蛋白质水平，异烟肼抗性不是由于第二位点突变(S)。对反式显性突变体的KatG蛋白的电泳分析表明，与WT相比，KatG二聚体减少，并形成活性降低的异源二聚体。导致这些缺陷的突变体聚集在建议的活性部位残基周围：N138S、T275P、S315T和D381G。为了确定可能界定参与亚基相互作用的KatG区域(S)的突变体，构建了C末端截断(带有和不带有D381G显性-负突变)。C端缺失既不能补充DeltakatG株，也不能对WT造成显性负效应。综上所述，这些结果表明KatG的氨基末端和羧基末端之间存在复杂的联系，可能与KatG二聚体形成的结构域交换机制一致。
In order to probe the structure and function of the mycobacterial catalase-peroxidase enzyme (KatG), we employed a genetic approach using dominant-negative analysis of katG merodiploids. Transformation of Mycobacterium bovis BCG with various katG point mutants (expressed from low-copy-number plasmids) resulted in reductions in peroxidase and catalase activities as measured in cell extracts. These reductions in enzymatic activity usually correlated with increased resistance to the antituberculosis drug isoniazid (INH). However, for the N138S trans-dominant mutant, the catalase-peroxidase activity was significantly decreased while the sensitivity to INH was retained. trans-dominance required katG expression from multicopy plasmids and could not be demonstrated with katG mutants integrated elsewhere on the wild-type M. bovis BCG chromosome. Reversal of the mutant phenotype through plasmid exchange suggested the catalase-peroxidase deficiency occurred at the protein level and that INH resistance was not due to a second site mutation(s). Electrophoretic analysis of KatG proteins from the trans-dominant mutants showed a reduction in KatG dimers compared to WT and formation of heterodimers with reduced activity. The mutants responsible for these defects cluster around proposed active site residues: N138S, T275P, S315T, and D381G. In an attempt to identify mutants that might delimit the region(s) of KatG involved in subunit interactions, C-terminal truncations were constructed (with and without the D381G dominant-negative mutation). None of the C-terminal deletions were able to complement a DeltakatG strain, nor could they cause a dominant-negative effect on the WT. Taken together, these results suggest an intricate association between the amino- and carboxyterminal regions of KatG and may be consistent with a domain-swapping mechanism for KatG dimer formation.