Mycobacterium tuberculosis MutT1 (Rv2985) and ADPRase (Rv1700) Proteins Constitute a Two-stage Mechanism of 8-Oxo-dGTP and 8-Oxo-GTP Detoxification and Adenosine to Cytidine Mutation Avoidance

Patil, Aravind Goud G.; Sang, Pau Biak; Govindan, Ashwin; Varshney, Umesh

doi:10.1074/jbc.m112.442566

Cited by 29 publications

(27 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Are there not other means of preventing or dealing with oxidized ribo lesions? Mycobacteria have MutT-type enzyme systems that can detoxify oxo-dGTP and oxo-rGTP in the NTP pool, by converting them to oxo-dGMP and oxo-rNMP (34,35). Whether the M. smegmatis MutT systems are operative in stationary phase at a level sufficient to cope with peroxide stress on the guanine nucleotide pool is not clear.…”

Section: Discussionmentioning

confidence: 99%

Division of labor amongMycobacterium smegmatisRNase H enzymes: RNase H1 activity of RnhA or RnhC is essential for growth whereas RnhB and RnhA guard against killing by hydrogen peroxide in stationary phase

et al. 2016

View full text Add to dashboard Cite

RNase H enzymes sense the presence of ribonucleotides in the genome and initiate their removal by incising the ribonucleotide-containing strand of an RNA:DNA hybrid. Mycobacterium smegmatis encodes four RNase H enzymes: RnhA, RnhB, RnhC and RnhD. Here, we interrogate the biochemical activity and nucleic acid substrate specificity of RnhA. We report that RnhA (like RnhC characterized previously) is an RNase H1-type magnesium-dependent endonuclease with stringent specificity for RNA:DNA hybrid duplexes. Whereas RnhA does not incise an embedded mono-ribonucleotide, it can efficiently cleave within tracts of four or more ribonucleotides in duplex DNA. We gained genetic insights to the division of labor among mycobacterial RNases H by deleting the rnhA, rnhB, rnhC and rnhD genes, individually and in various combinations. The salient conclusions are that: (i) RNase H1 activity is essential for mycobacterial growth and can be provided by either RnhC or RnhA; (ii) the RNase H2 enzymes RnhB and RnhD are dispensable for growth and (iii) RnhB and RnhA collaborate to protect M. smegmatis against oxidative damage in stationary phase. Our findings highlight RnhC, the sole RNase H1 in pathogenic mycobacteria, as a candidate drug discovery target for tuberculosis and leprosy.

show abstract

Section: Discussionmentioning

confidence: 99%

Division of labor amongMycobacterium smegmatisRNase H enzymes: RNase H1 activity of RnhA or RnhC is essential for growth whereas RnhB and RnhA guard against killing by hydrogen peroxide in stationary phase

et al. 2016

View full text Add to dashboard Cite

show abstract

“…An example of a predicted phenotype from a rescue experiment is the expression of mutT1 from Mycobacterium tuberculosis in mutT deficient E. coli . The mutT1 rescued E. coli by reducing the A:T → C:G mutation rate . Finally, the maximum between S knockout/knockdown and S rescue was taken as the value of S genetic .…”

Section: Methodsmentioning

confidence: 99%

The evolution of function within the Nudix homology clan

Srouji

Park

et al. 2017

Proteins

View full text Add to dashboard Cite

The Nudix homology clan encompasses over 80,000 protein domains from all three domains of life, defined by homology to each other. Proteins with a domain from this clan fall into four general functional classes: pyrophosphohydrolases, isopentenyl diphosphate isomerases (IDIs), adenine/guanine mismatch-specific adenine glycosylases (A/G-specific adenine glycosylases), and non-enzymatic activities such as protein/protein interaction and transcriptional regulation. The largest group, pyrophosphohydrolases, encompasses more than 100 distinct hydrolase specificities. To understand the evolution of this vast number of activities, we assembled and analyzed experimental and structural data for 205 Nudix proteins collected from the literature. We corrected erroneous functions or provided more appropriate descriptions for 53 annotations described in the Gene Ontology Annotation database in this family, and propose 275 new experimentally-based annotations. We manually constructed a structure-guided sequence alignment of 78 Nudix proteins. Using the structural alignment as a seed, we then made an alignment of 347 “select” Nudix homology domains, curated from structurally determined, functionally characterized, or phylogenetically important Nudix domains. Based on our review of Nudix pyrophosphohydrolase structures and specificities, we further analyzed a loop region downstream of the Nudix hydrolase motif previously shown to contact the substrate molecule and possess known functional motifs. This loop region provides a potential structural basis for the functional radiation and evolution of substrate specificity within the hydrolase family. Finally, phylogenetic analyses of the 347 select protein domains and of the complete Nudix homology clan revealed general monophyly with regard to function and a few instances of probable homoplasy.

show abstract

“…Patil et al measured the K m and k cat of Rv1700 for the hydrolysis of 8-oxo-2′-deoxyguanosine -5′-diphosphate (8-oxo-dGDP), producing 8-oxo-dGMP and a free phosphate. Interestingly, the catalytic efficiency ( k cat /K m ) of Rv1700 is much higher for the hydrolysis of ADPR than 8-oxo-dGDP, suggesting a more relevant biological role of Rv1700 in hydrolysis of ADPR (The k cat / K m of ADPR is 14.4 s -1 /200 μM= 0.072 in the presence of Mg 2+ ; k cat / K m of 8-oxo-dGTP is 0.0164 s -1 /9.5 μM= 0.0017) (Eisenthal et al 2007; Patil et al 2013). …”

Section: Resultsmentioning

confidence: 99%

Kinetic and mutational studies of the adenosine diphosphate ribose hydrolase from Mycobacterium tuberculosis

O'Handley

Thirawatananond

Kang

et al. 2016

J Bioenerg Biomembr

View full text Add to dashboard Cite

Mycobacterium tuberculosis represents one of the world's most devastating infectious agents – with one third of the world's population infected and 1.5 million people dying each year from this deadly pathogen. As part of an effort to identify targets for therapeutic intervention, we carried out the kinetic characterization of the product of gene rv1700 of M tuberculosis. Based on its sequence and its structure, the protein had been tentatively identified as a pyrophosphohydrolase specific for adenosine diphosphate ribose (ADPR), a compound involved in various pathways including oxidative stress response and tellurite resistance. In this work we carry out a kinetic, mutational and structural investigation of the enzyme, which provides a full characterization of this Mt-ADPRase. Optimal catalytic rates were achieved at alkaline pH (7.5-8.5) with either 0.5-1 mM Mg2+ or 0.02-1 mM Mn2+. Km and kcat values for hydrolysis of ADPR with Mg2+ ions are 200±19 μM and 14.4±0.4 s-1, and with Mn2+ ions are 554±64 μM and 28.9±1.4 s-1. Four residues proposed to be important in the catalytic mechanism of the enzyme were individually mutated and the kinetics of the mutant enzymes were characterized. In the four cases, the Km increased only slightly (2- to 3-fold) but the kcat decreased significantly (300- to 1900-fold), confirming the participation of these residues in catalysis. An analysis of the sequence and structure conservation patterns in Nudix ADPRases permits an unambiguous identification of members of the family and provides insight into residues involved in catalysis and their participation in substrate recognition in the Mt-ADPRase.

show abstract

Mycobacterium tuberculosis MutT1 (Rv2985) and ADPRase (Rv1700) Proteins Constitute a Two-stage Mechanism of 8-Oxo-dGTP and 8-Oxo-GTP Detoxification and Adenosine to Cytidine Mutation Avoidance

Cited by 29 publications

References 32 publications

Division of labor amongMycobacterium smegmatisRNase H enzymes: RNase H1 activity of RnhA or RnhC is essential for growth whereas RnhB and RnhA guard against killing by hydrogen peroxide in stationary phase

Division of labor amongMycobacterium smegmatisRNase H enzymes: RNase H1 activity of RnhA or RnhC is essential for growth whereas RnhB and RnhA guard against killing by hydrogen peroxide in stationary phase

The evolution of function within the Nudix homology clan

Kinetic and mutational studies of the adenosine diphosphate ribose hydrolase from Mycobacterium tuberculosis

Contact Info

Product

Resources

About