甲醛的积累,高反应性分子是细胞毒性的,必须被消除才能使生物体存活。甲醛解毒系统存在于几乎所有的生物体中,包括致病性和非致病性分枝杆菌。MscR,来自耻垢分枝杆菌(Msm)的甲醛脱氢酶,是该系统不可或缺的一部分,并与其下游未表征的基因形成双顺反子操纵子,fmh.我们在这里展示Fmh,一种推定的金属-β-内酰胺酶,在体内与mscR共同过表达时,对耐受较高量的甲醛至关重要。我们的核磁共振研究表明,MscR,随着FMH,通过Mymothiol(MSH)依赖性途径增强甲酸盐的产生,强调Fmh在甲醛解毒中的重要性。虽然另一种醛脱氢酶,MSMEG_1543,在甲醛添加后诱导,它没有参与排毒。我们还表明,mscR操纵子的表达是组成型的,并且在添加甲醛时保持不变,如PmscR的启动子活性以及MscR的转录物和蛋白质水平所显示的。此外,我们建立了巯基反应sigma因子SigH在甲醛解毒中的作用。我们证明SigH,而不是SigE,对甲醛解毒至关重要,即使它不直接调节mscR操纵子的表达。此外,sigH敲除中对甲醛的敏感性可以通过mscR的过表达来减轻。一起来看,我们的数据证明了MSH依赖性途径在分枝杆菌系统中甲醛解毒的重要性.真核生物中缺乏这种MSH依赖性蛋白质,并且在结核分枝杆菌中完全保守,结核病的病原体,进一步解开这种病原体的新药物靶标。重要的是已经对不同细菌中的甲醛解毒进行了广泛的研究。然而,我们目前对分枝杆菌这一过程的潜在机制的理解仍然很少。我们之前证明了MscR,一种来自耻垢分枝杆菌的甲醛脱氢酶,在这种解毒途径中起着关键作用。这里,我们提出了一种潜在的S-甲酰-分枝杆菌硫醇水解酶,名为Fmh,被认为是金属β-内酰胺酶,它与分枝杆菌硫醇(MSH)和MscR一起发挥作用,以增强该解毒途径中的甲酸生产。Fmh与MscR的共表达显着提高了耻垢分枝杆菌的甲醛解毒效率。我们的实验确定Fmh催化该解毒途径的最后一步。尽管发现另一种sigma因子SigH参与甲醛解毒,它不直接调节mscR的表达。由于甲醛解毒对细菌生存至关重要,我们预计这一过程将成为根除结核分枝杆菌的潜在药物靶点.
The build-up of formaldehyde, a highly reactive molecule is cytotoxic and must be eliminated for the organism\'s survival. Formaldehyde detoxification system is found in nearly all organisms including both pathogenic and non-pathogenic mycobacteria. MscR, a formaldehyde dehydrogenase from Mycobacterium smegmatis (Msm), is an indispensable part of this system and forms a bicistronic operon with its downstream uncharacterized gene, fmh. We here show that Fmh, a putative metallo-beta-lactamase, is essential in tolerating higher amounts of formaldehyde when co-overexpressed with mscR in vivo. Our NMR studies indicate that MscR, along with Fmh, enhances formate production through a mycothiol (MSH)-dependent pathway, emphasizing the importance of Fmh in detoxifying formaldehyde. Although another aldehyde dehydrogenase, MSMEG_1543, induces upon formaldehyde addition, it is not involved in its detoxification. We also show that the expression of the mscR operon is constitutive and remains unchanged upon formaldehyde addition, as displayed by the promoter activity of PmscR and by the transcript and protein levels of MscR. Furthermore, we establish the role of a thiol-responsive sigma factor SigH in formaldehyde detoxification. We show that SigH, and not SigE, is crucial for formaldehyde detoxification, even though it does not directly regulate mscR operon expression. In addition, sensitivity to formaldehyde in sigH-knockout could be alleviated by overexpression of mscR. Taken together, our data demonstrate the importance of MSH-dependent pathways in detoxifying formaldehyde in a mycobacterial system. An absence of such MSH-dependent proteins in eukaryotes and its complete conservation in M. tuberculosis, the causative agent of tuberculosis, further unravel new drug targets for this pathogen.IMPORTANCEExtensive research has been done on formaldehyde detoxification in different bacteria. However, our current understanding of the mechanisms underlying this process in mycobacteria remains exceedingly little. We previously showed that MscR, a formaldehyde dehydrogenase from Mycobacterium smegmatis, plays a pivotal role in this detoxification pathway. Here, we present a potential S-formyl-mycothiol hydrolase named Fmh, thought to be a metallo-beta-lactamase, which functions along with mycothiol (MSH) and MscR to enhance formate production within this detoxification pathway. Co-expression of Fmh with MscR significantly enhances the efficiency of formaldehyde detoxification in M. smegmatis. Our experiments establish that Fmh catalyzes the final step of this detoxification pathway. Although an alternative sigma factor SigH was found to be involved in formaldehyde detoxification, it did not directly regulate the expression of mscR. Since formaldehyde detoxification is essential for bacterial survival, we envisage this process to be a potential drug target for M. tuberculosis eradication.