Characterization of the catalytic signature of Scabin toxin, a DNA-targeting ADP-ribosyltransferase

2018 ◽  
Vol 475 (1) ◽  
pp. 225-245 ◽  
Author(s):  
Bronwyn Lyons ◽  
Miguel R. Lugo ◽  
Stephanie Carlin ◽  
Taylor Lidster ◽  
A. Rod Merrill
Keyword(s):  
Reaction Mechanism ◽  
Dna Binding ◽  
Molecular Model ◽  
Competitive Inhibitor ◽  
Binding Motif ◽  
Transferase Activity ◽  
Dna Targeting ◽  
Key Residues ◽  
Adp Ribosyltransferase

Scabin was previously identified as a novel DNA-targeting mono-ADP-ribosyltransferase (mART) toxin from the plant pathogen 87.22 strain of Streptomyces scabies. Scabin is a member of the Pierisin-like subgroup of mART toxins, since it targets DNA. An in-depth characterization of both the glycohydrolase and transferase enzymatic activities of Scabin was conducted. Several protein variants were developed based on an initial Scabin·DNA molecular model. Consequently, three residues were deemed important for DNA-binding and transferase activity. Trp128 and Trp155 are important for binding the DNA substrate and participate in the reaction mechanism, whereas Tyr129 was shown to be important only for DNA binding, but was not involved in the reaction mechanism. Trp128 and Trp155 are both conserved within the Pierisin-like toxins, whereas Tyr129 is a unique substitution within the group. Scabin showed substrate specificity toward double-stranded DNA containing a single-base overhang, as a model for single-stranded nicked DNA. The crystal structure of Scabin bound to NADH — a competitive inhibitor of Scabin — was determined, providing important insights into the active-site structure and Michaelis–Menten complex of the enzyme. Based on these results, a novel DNA-binding motif is proposed for Scabin with substrate and the key residues that may participate in the Scabin·NAD(+) complex are highlighted.

scholarly journals Molecular characterization of a glycosylphosphatidylinositol-linked ADP- ribosyltransferase from lymphocytes

Blood ◽  
1996 ◽  
Vol 88 (3) ◽  
pp. 915-921 ◽  
Author(s):  
IJ Okazaki ◽  
HJ Kim ◽  
NG McElvaney ◽  
E Lesma ◽  
J Moss
Keyword(s):  
Transfer Reaction ◽  
Mammary Adenocarcinoma ◽  
Bacterial Toxin ◽  
Common Mechanism ◽  
Transferase Activity ◽  
Inhibitory Effects ◽  
Regulation Of Proliferation ◽  
Adp Ribosyltransferase ◽  
Ribose Moiety

Abstract Mono ADP-ribosyltransferases catalyze the transfer of the ADP-ribose moiety of nicotinamide adenine dinucleotide (NAD) to proteins. It was reported by Wang et al (J Immunol 153:4048, 1994) that incubation of mouse cytotoxic T lymphocytes (CTL) with NAD resulted in the ADP- ribosylation of membrane proteins and inhibition of cell proliferation and cytotoxicity. Treatment of CTL with phosphatidylinositol-specific phospholipase C (PI-PLC) before incubation with NAD prevented the inhibitory effects of NAD on the cells, consistent with the removal of a glycosylphosphatidylinositol (GPI)-anchored ADP-ribosyltransferase on the lymphocyte surface. We have identified and cloned a GPI-linked ADP- ribosyltransferase from Yac-1 mouse T-cell lymphoma cells. The deduced amino acid sequence of the Yac-1 transferase was 70% and 41% identical to those of the rabbit skeletal muscle and chicken heterophil, respectively. It contained three noncontiguous sequences similar to those found in several of the bacterial toxin and vertebrate ADP- ribosyltransferases. Based on crystallography of the bacterial toxins, these regions are believed to form, in part, the catalytic site consistent with a common mechanism for the ADP-ribose transfer reaction. In rat mammary adenocarcinoma (NMU) cells transformed with the Yac-1 transferase cDNA, transferase activity was present on the cell surface and was released into the medium by treatment of cells with PI-PLC. Thus, we have cloned a novel gene that has properties identical to the transferase detected in CTL, and may be involved in the NAD-dependent regulation of proliferation and cytotoxicity.

scholarly journals Mapping the DNA-Binding Motif of Scabin Toxin, a Guanine Modifying Enzyme from Streptomyces scabies

Toxins ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 55
Author(s):  
Maritza Vatta ◽  
Bronwyn Lyons ◽  
Kayla A. Heney ◽  
Taylor Lidster ◽  
A. Rod Merrill
Keyword(s):  
Dna Binding ◽  
Molecular Model ◽  
Md Simulations ◽  
Streptomyces Scabies ◽  
Binding Motif ◽  
Sequence Motif ◽  
Arginine Residues ◽  
Guanine Base ◽  
Dna Substrates ◽  
Dna Substrate

Scabin is a mono-ADP-ribosyltransferase toxin/enzyme and possible virulence factor produced by the agriculture pathogen, Streptomyces scabies. Recently, molecular dynamic approaches and MD simulations revealed its interaction with both NAD+ and DNA substrates. An Essential Dynamics Analysis identified a crab-claw-like mechanism, including coupled changes in the exposed motifs, and the Rβ1-RLa-NLc-STTβ2-WPN-WARTT-(QxE)ARTT sequence motif was proposed as a catalytic signature of the Pierisin family of DNA-acting toxins. A new fluorescence assay was devised to measure the kinetics for both RNA and DNA substrates. Several protein variants were prepared to probe the Scabin-NAD-DNA molecular model and to reveal the reaction mechanism for the transfer of ADP-ribose to the guanine base in the DNA substrate. The results revealed that there are several lysine and arginine residues in Scabin that are important for binding the DNA substrate; also, key residues such as Asn110 in the mechanism of ADP-ribose transfer to the guanine base were identified. The DNA-binding residues are shared with ScARP from Streptomyces coelicolor but are not conserved with Pierisin-1, suggesting that the modification of guanine bases by ADP-ribosyltransferases is divergent even in the Pierisin family.

scholarly journals Molecular characterization of a glycosylphosphatidylinositol-linked ADP- ribosyltransferase from lymphocytes

Blood ◽  
1996 ◽  
Vol 88 (3) ◽  
pp. 915-921 ◽  
Author(s):  
IJ Okazaki ◽  
HJ Kim ◽  
NG McElvaney ◽  
E Lesma ◽  
J Moss
Keyword(s):  
Transfer Reaction ◽  
Mammary Adenocarcinoma ◽  
Bacterial Toxin ◽  
Common Mechanism ◽  
Transferase Activity ◽  
Inhibitory Effects ◽  
Regulation Of Proliferation ◽  
Adp Ribosyltransferase ◽  
Ribose Moiety

Mono ADP-ribosyltransferases catalyze the transfer of the ADP-ribose moiety of nicotinamide adenine dinucleotide (NAD) to proteins. It was reported by Wang et al (J Immunol 153:4048, 1994) that incubation of mouse cytotoxic T lymphocytes (CTL) with NAD resulted in the ADP- ribosylation of membrane proteins and inhibition of cell proliferation and cytotoxicity. Treatment of CTL with phosphatidylinositol-specific phospholipase C (PI-PLC) before incubation with NAD prevented the inhibitory effects of NAD on the cells, consistent with the removal of a glycosylphosphatidylinositol (GPI)-anchored ADP-ribosyltransferase on the lymphocyte surface. We have identified and cloned a GPI-linked ADP- ribosyltransferase from Yac-1 mouse T-cell lymphoma cells. The deduced amino acid sequence of the Yac-1 transferase was 70% and 41% identical to those of the rabbit skeletal muscle and chicken heterophil, respectively. It contained three noncontiguous sequences similar to those found in several of the bacterial toxin and vertebrate ADP- ribosyltransferases. Based on crystallography of the bacterial toxins, these regions are believed to form, in part, the catalytic site consistent with a common mechanism for the ADP-ribose transfer reaction. In rat mammary adenocarcinoma (NMU) cells transformed with the Yac-1 transferase cDNA, transferase activity was present on the cell surface and was released into the medium by treatment of cells with PI-PLC. Thus, we have cloned a novel gene that has properties identical to the transferase detected in CTL, and may be involved in the NAD-dependent regulation of proliferation and cytotoxicity.

scholarly journals Structural and biochemical characterization of an RNA/DNA binding motif in the N-terminal domain of RecQ4 helicases

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Francesca Marino ◽  
Aditya Mojumdar ◽  
Chiara Zucchelli ◽  
Amit Bhardwaj ◽  
Emanuele Buratti ◽  
...  
Keyword(s):  
Dna Binding ◽  
Biochemical Characterization ◽  
Binding Motif ◽  
Terminal Domain

scholarly journals Real-time tracking reveals the catalytic process of Rad51-driven DNA strand exchange

2019 ◽  
Author(s):  
Kentaro Ito ◽  
Yasuto Murayama ◽  
Yumiko Kurokawa ◽  
Shuji Kanamaru ◽  
Yuichi Kokabu ◽  
...  
Keyword(s):  
Dna Binding ◽  
Molecular Model ◽  
Strand Exchange ◽  
Catalytic Process ◽  
Binding Motif ◽  
Binding Motifs ◽  
Dna Binding Motifs ◽  
Dna Strand Exchange ◽  
Motif Site ◽  
Dna Strand

AbstractDuring homologous recombination, Rad51 forms a nucleoprotein filament on single-stranded DNA to promote DNA strand exchange. This filament binds to double-stranded DNA (dsDNA), searches for homology, and promotes transfer of the complementary strand, producing a new heteroduplex. Strand exchange proceeds via two distinct three-strand intermediates, C1 and C2. C1 contains the intact donor dsDNA whereas C2 contains newly formed heteroduplex DNA. Here, we show that conserved DNA binding motifs, loop 1 (L1) and loop 2 (L2) in site I of Rad51, play distinct roles in this process. L1 is involved in formation of the C1 complex whereas L2 mediates the C1-C2 transition, producing the heteroduplex. Another DNA binding motif, site II, serves as the DNA entry position for initial Rad51 filament formation, as well as for second donor dsDNA incorporation. Our study provides a comprehensive molecular model for the catalytic process of strand exchange mediated by eukaryotic RecA family recombinases.

Targeting IDH Mutations in AML: Wielding the Double-edged Sword of Differentiation

2020 ◽  
Vol 20 (7) ◽  
pp. 490-500 ◽  
Author(s):  
Justin S. Becker ◽  
Amir T. Fathi
Keyword(s):  
Genome Structure ◽  
Cellular Metabolism ◽  
Standard Of Care ◽  
Competitive Inhibitor ◽  
Driver Mutations ◽  
New Class ◽  
Regulatory Enzymes ◽  
Idh Mutations ◽  
Genomic Characterization

The genomic characterization of acute myeloid leukemia (AML) by DNA sequencing has illuminated subclasses of the disease, with distinct driver mutations, that might be responsive to targeted therapies. Approximately 15-23% of AML genomes harbor mutations in one of two isoforms of isocitrate dehydrogenase (IDH1 or IDH2). These enzymes are constitutive mediators of basic cellular metabolism, but their mutated forms in cancer synthesize an abnormal metabolite, 2- hydroxyglutarate, that in turn acts as a competitive inhibitor of multiple gene regulatory enzymes. As a result, leukemic IDH mutations cause changes in genome structure and gene activity, culminating in an arrest of normal myeloid differentiation. These discoveries have motivated the development of a new class of selective small molecules with the ability to inhibit the mutant IDH enzymes while sparing normal cellular metabolism. These agents have shown promising anti-leukemic activity in animal models and early clinical trials, and are now entering Phase 3 study. This review will focus on the growing preclinical and clinical data evaluating IDH inhibitors for the treatment of IDH-mutated AML. These data suggest that inducing cellular differentiation is central to the mechanism of clinical efficacy for IDH inhibitors, while also mediating toxicity for patients who experience IDH Differentiation Syndrome. Ongoing trials are studying the efficacy of IDH inhibitors in combination with other AML therapies, both to evaluate potential synergistic combinations as well as to identify the appropriate place for IDH inhibitors within existing standard-of-care regimens.

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