Analysis of human ITPase nucleobase specificity by site-directed mutagenesis

Gall, Amanda D.; Gall, Anthony; Moore, Ashley C.; Aune, Martin K.; Heid, Steven; Mori, Ayaka; Burgis, Nicholas E.

doi:10.1016/j.biochi.2013.05.016

Cited by 13 publications

(43 citation statements)

References 34 publications

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“…This mutation is predicted to alter electrostatic interactions of the CBSV Ham1 protein and thereby affect function. Gall et al ., () previously demonstrated that mutating the SHR motif to SAA in the human ITPase protein abolishes ITP substrate specificity or activity. Second, to also test whether the CBSV_Tanza and UCBSV_Kikombe Ham1 proteins are associated with differential symptom development during N. benthamiana infections, the chimeric CBSV_UHam IC was constructed, consisting of the CBSV_Tanza genome with a UCBSV_Kikombe Ham1 sequence replacement (Fig.…”

Section: Resultsmentioning

confidence: 98%

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Cassava brown streak virus Ham1 protein hydrolyses mutagenic nucleotides and is a necrosis determinant

Tomlinson

Pablo‐Rodriguez

Bunawan

et al. 2019

Molecular Plant Pathology

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Summary Cassava brown streak disease (CBSD) is a leading cause of cassava losses in East and Central Africa, and is currently having a severe impact on food security. The disease is caused by two viruses within the Potyviridae family: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), which both encode atypical Ham1 proteins with highly conserved inosine triphosphate (ITP) pyrophosphohydrolase (ITPase) domains. ITPase proteins are widely encoded by plant, animal, and archaea. They selectively hydrolyse mutagenic nucleotide triphosphates to prevent their incorporation into nucleic acid and thereby function to reduce mutation rates. It has previously been hypothesized that U/CBSVs encode Ham1 proteins with ITPase activity to reduce viral mutation rates during infection. In this study, we investigate the potential roles of U/CBSV Ham1 proteins. We show that both CBSV and UCBSV Ham1 proteins have ITPase activities through in vitro enzyme assays. Deep‐sequencing experiments found no evidence of the U/CBSV Ham1 proteins providing mutagenic protection during infections of Nicotiana hosts. Manipulations of the CBSV_Tanza infectious clone were performed, including a Ham1 deletion, ITPase point mutations, and UCBSV Ham1 chimera. Unlike severely necrotic wild‐type CBSV_Tanza infections, infections of Nicotiana benthamiana with the manipulated CBSV infectious clones do not develop necrosis, indicating that that the CBSV Ham1 is a necrosis determinant. We propose that the presence of U/CBSV Ham1 proteins with highly conserved ITPase motifs indicates that they serve highly selectable functions during infections of cassava and may represent a euphorbia host adaptation that could be targeted in antiviral strategies.

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Section: Resultsmentioning

confidence: 98%

“…This prevents XTP/ITP incorporation into nucleic acid and thereby reduces mutation rates. U/CBSV Ham1 proteins contain the highly conserved serine‐histidine‐arginine (SHR) signature motif, which in the human ITPase is involved with substrate binding and specificity (Gall et al , ; Stenmark et al , ).…”

Section: Introductionmentioning

confidence: 99%

Cassava brown streak virus Ham1 protein hydrolyses mutagenic nucleotides and is a necrosis determinant

Tomlinson

Pablo‐Rodriguez

Bunawan

et al. 2019

Molecular Plant Pathology

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“…This structure also revealed specific recognition of the 2-keto oxygen of ITP, with a hydrogen bond and explained the enzyme reactivity differences between inosine and guanosine nucleotides. The role of the major residue determinants that were considered to be responsible for the specific recognition or steric exclusion of the respective purine bases was later also confirmed by site-directed mutagenesis [80]. A model for the catalytic mechanism was provided based on structural insights from the ITPase homologue RgdB [35].…”

Section: All-b Dutpasementioning

confidence: 94%

“…A model for the catalytic mechanism was provided based on structural insights from the ITPase homologue RgdB [35]. The role of a conserved Asp as a general base, coordinating and deprotonating the catalytic nucleophile for in-line nucleophilic attack on the a-phosphate, was confirmed by structural analyses (PDB code: 2Q16) ( Figs 3B and 4A), as well as by site-directed mutagenesis [35,78,80]. Post-catalytic deprotonation of the general base after completion of hydrolysis was suggested to be performed by the nearby Lys53.…”

Section: Enzymementioning

confidence: 94%

Preventive DNA repair by sanitizing the cellular (deoxy)nucleoside triphosphate pool

2014

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The occurrence of modified bases in DNA is attributed to some major factors: incorporation of altered nucleotide building blocks and chemical reactions or radiation effects on bases within the DNA structure. Several enzyme families are involved in preventing the incorporation of noncanonical bases playing a 'sanitizing' role. The catalytic mechanism of action of these enzymes has been revealed for a number of representatives in clear structural and kinetic detail. In this review, we focus in detail on those examples where clear evidence has been produced using high-resolution structural studies. Comparing the protein fold and architecture of the enzyme active sites, two main classes of sanitizing deoxyribonucleoside triphosphate pyrophosphatases can be assigned that are distinguished by the site of nucleophilic attack. In enzymes associated with attack at the a-phosphorus, it is shown that coordination of the c-phosphate group is also ensured by multiple interactions. By contrast, enzymes catalyzing attack at the b-phosphorus atom mainly coordinate the a-and the b-phosphate only. Characteristic differences are also observed with respect to the role of the metal ion cofactor (Mg 2+ ) and the coordination of nucleophilic water. Using different catalytic mechanisms embedded in different protein folds, these enzymes present a clear example of convergent evolution.

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“…In 2015, Kevelam et al demonstrated that arginine to cysteine substitution at position 178 (R178C (c.532C > T)) produced an ITPA deficient phenotype which resulted in a lethal early infantile encephalopathy (Kevelam et al, 2015). This position has previously been identified as essential for enzyme activity based on a site-directed mutational analysis of the substratebinding site (Gall et al, 2013). ITPA also plays a role in drug metabolism and lower levels of ITPA activity can modulate drug metabolism for some treatment regimens.…”

Section: Introductionmentioning

confidence: 99%

Structural dynamics of inosine triphosphate pyrophosphatase (ITPA) protein and two clinically relevant mutants: molecular dynamics simulations

Houndonougbo

Pugh

VanWormer

et al. 2020

Journal of Biomolecular Structure and Dynamics

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The inosine triphosphate pyrophosphatase (ITPA) protein is responsible for removing noncanonical purine nucleoside triphosphates from intracellular nucleotide pools. Absence of ITPA results in genomic instability and increased levels of inosine in DNA and RNA. The proline to threonine substitution at position 32 (P32T) affects roughly 15% of the global population and can modulate treatment outcomes for cancer, lupus, and hepatitis C patients. The substitution of arginine with cysteine at position 178 (R178C) is extremely uncommon and has only been reported in a small cohort of early infantile encephalopathy patients suggesting that a functional ITPA protein is required for life in humans. Here we present molecular dynamic simulations that describe the structure and dynamics of the wild-type ITPA homodimer and two of its clinically relevant mutants, P32T and R178C. The simulation results indicate that both the P32T and R178C mutations alter the structure and dynamic properties of the protein and provide a possible explanation of the experimentally observed effect of the mutations on ITPA activity. Specifically, the mutations increased the overall flexibility of the protein and changed the dominant collective motions of the top lobe as well as the helix 2 of the lower lobe. Moreover, we have identified key active-site residues that are classified as essential or intermediate for inosine triphosphate (ITP) hydrolyzing activity based on their hydrogen bond occupancy. Here we also present biochemical data indicating that the R178C mutant has very low ITP hydrolyzing activity. ARTICLE HISTORY

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Analysis of human ITPase nucleobase specificity by site-directed mutagenesis

Cited by 13 publications

References 34 publications

Cassava brown streak virus Ham1 protein hydrolyses mutagenic nucleotides and is a necrosis determinant

Cassava brown streak virus Ham1 protein hydrolyses mutagenic nucleotides and is a necrosis determinant

Preventive DNA repair by sanitizing the cellular (deoxy)nucleoside triphosphate pool

Structural dynamics of inosine triphosphate pyrophosphatase (ITPA) protein and two clinically relevant mutants: molecular dynamics simulations

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