Abstract
Lack of molybdenum cofactor (Moco) in Escherichia coli leads to hypersensitivity to the mutagenic and toxic effects of N-hydroxylated base analogs, such as 6-N-hydroxylaminopurine (HAP). This phenotype is due to the loss of two Moco-dependent activities, YcbX and YiiM, that are capable of reducing HAP to adenine. Here, we describe two novel HAP-sensitive mutants containing a defect in iscS or tusA (yhhP) gene. IscS is a major L-cysteine desulfurase involved in iron–sulfur cluster synthesis, thiamine synthesis, and tRNA thiomodification. TusA is a small sulfur-carrier protein that interacts with IscS. We show that both IscS and TusA operate within the Moco-dependent pathway. Like other Moco-deficient strains, tusA and iscS mutants are HAP sensitive and resistant to chlorate under anaerobic conditions. The base-analog sensitivity of iscS or tusA strains could be suppressed by supplying exogenous L-cysteine or sulfide or by an increase in endogenous sulfur donors (cysB constitutive mutant). The data suggest that iscS and tusA mutants have a defect in the mobilization of sulfur required for active YcbX/YiiM proteins as well as nitrate reductase, presumably due to lack of functional Moco. Overall, our data imply a novel and indispensable role of the IscS/TusA complex in the activity of several molybdoenzymes.
Highlights
Base analogs are modified nucleobases that can substitute for the natural bases in cellular metabolism, and their involvement can lead to toxic and/or mutagenic consequences
TusA encodes an 81-amino acid polypeptide that physically interacts with IscS. The latter is an L-cysteine desulfurase that is a key enzyme in the biosynthesis in iron–sulfur clusters and some other sulfur-related activities (Fontecave et al 2008; Roche et al 2013)
In our efforts to understand the mechanisms by which cells can detoxify mutagenic and toxic chemicals such as N-hydroxylated compounds we have previously characterized novel activities that depended on at least two proteins, YcbX and YiiM, both requiring the molybdenum cofactor (Moco)
Summary
Base analogs are modified nucleobases that can substitute for the natural bases in cellular metabolism, and their involvement can lead to toxic and/or mutagenic consequences. Our previous studies have shown that Escherichia coli strains lacking molybdenum cofactor (Moco) are hypersensitive to the toxic and mutagenic action of HAP (Fig. 1A) and related N-hydroxylated analogs, including AHAP, HC, and hydroxylamine (NH2OH) (Kozmin et al 2000; Kozmin and Schaaper 2007). We demonstrated that Moco-dependent base-analog detoxification is due to action of two Moco-dependent enzymes, YcbX and YiiM, which were shown capable of reducing HAP to nontoxic adenine (Kozmin et al 2008). As part of a continuing investigation of the cellular factors that determine resistance to toxic N-hydroxylated compounds, and to better understand the regulation of Moco and Moco-dependent enzymes, we have undertaken further searches for genetic mutants of E. coli that display altered sensitivity to the base-analog HAP. Our results reveal a novel correlation between the activity of Moco-dependent enzymes and cellular sulfur metabolism
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