scholarly journals Roles of the hydroxy group of tyrosine in crystal structures of Sulfurisphaera tokodaii O 6-methylguanine-DNA methyltransferase

2021 ◽  
Vol 77 (12) ◽  
Author(s):  
Makiko Kikuchi ◽  
Takahiro Yamauchi ◽  
Yasuhito Iizuka ◽  
Masaru Tsunoda
Keyword(s):  
Crystal Structure ◽  
Crystal Structures ◽  
Hydroxy Group ◽  
Active Site ◽  
Dna Methyltransferase ◽  
Structural Changes ◽  
Molecular Mechanisms ◽  
Alkylating Agents ◽  
Substrate Analogs ◽  
Methylguanine Dna Methyltransferase

O 6-Methylguanine-DNA methyltransferase (MGMT) removes cytotoxic O 6-alkyl adducts on the guanine base and protects the cell from genomic damage induced by alkylating agents. Although there are reports of computational studies on the activity of the enzyme with mutations at tyrosine residues, no studies concerning the crystal structure of its mutants have been found. In this study, the function of Tyr91 was investigated in detail by comparing the crystal structures of mutants and their complexes with substrate analogs. In this study, tyrosine, a conserved amino acid near the active-site loop in the C-terminal domain of Sulfurisphaera tokodaii MGMT (StoMGMT), was mutated to phenylalanine to produce a Y91F mutant, and the cysteine which is responsible for receiving the methyl group in the active site was mutated to a serine to produce a C120S mutant. A Y91F/C120S double-mutant StoMGMT was also created. The function of tyrosine is discussed based on the crystal structure of Y91F mutant StoMGMT. The crystal structures of StoMGMT were determined at resolutions of 1.13–2.60 Å. They showed no structural changes except in the mutated part. No electron density for deoxyguanosine or methyl groups was observed in the structure of Y91F mutant crystals immersed in O 6-methyl-2′-deoxyguanosine, nor was the group oxidized in wild-type StoMGMT. Therefore, the hydroxy group of Tyr91 may prevent the oxidant from entering the active site. This suggests that tyrosine, which is highly conserved at the N-terminus of the helix–turn–helix motif across species, protects the active site of MGMTs, which are deactivated after repairing only one alkyl adduct. Overall, the results may provide a basis for understanding the molecular mechanisms by which high levels of conserved amino acids play a role in ensuring the integrity of suicide enzymes, in addition to promoting their activity.

scholarly journals How cyanophage S-2L rejects adenine and incorporates 2-aminoadenine to saturate hydrogen bonding in its DNA

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Dariusz Czernecki ◽  
Pierre Legrand ◽  
Mustafa Tekpinar ◽  
Sandrine Rosario ◽  
Pierre-Alexandre Kaminski ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Hydrogen Bonding ◽  
Crystal Structures ◽  
Active Site ◽  
Metal Ion ◽  
Restriction Endonucleases ◽  
Structural Element

AbstractBacteriophages have long been known to use modified bases in their DNA to prevent cleavage by the host’s restriction endonucleases. Among them, cyanophage S-2L is unique because its genome has all its adenines (A) systematically replaced by 2-aminoadenines (Z). Here, we identify a member of the PrimPol family as the sole possible polymerase of S-2L and we find it can incorporate both A and Z in front of a T. Its crystal structure at 1.5 Å resolution confirms that there is no structural element in the active site that could lead to the rejection of A in front of T. To resolve this contradiction, we show that a nearby gene is a triphosphohydolase specific of dATP (DatZ), that leaves intact all other dNTPs, including dZTP. This explains the absence of A in S-2L genome. Crystal structures of DatZ with various ligands, including one at sub-angstrom resolution, allow to describe its mechanism as a typical two-metal-ion mechanism and to set the stage for its engineering.

scholarly journals In Silico Prediction of O6-Methylguanine-DNA Methyltransferase Inhibitory Potency of Base Analogs with QSAR and Machine Learning Methods

Molecules ◽  
2018 ◽  
Vol 23 (11) ◽  
pp. 2892 ◽  
Author(s):  
Guohui Sun ◽  
Tengjiao Fan ◽  
Xiaodong Sun ◽  
Yuxing Hao ◽  
Xin Cui ◽  
...  
Keyword(s):  
Machine Learning ◽  
Dna Methyltransferase ◽  
Information Gain ◽  
Alkylating Agents ◽  
External Validation ◽  
Learning Methods ◽  
Methylguanine Dna Methyltransferase ◽  
Machine Learning Methods ◽  
Dragon Descriptors ◽  
External Test

O6-methylguanine-DNA methyltransferase (MGMT), a unique DNA repair enzyme, can confer resistance to DNA anticancer alkylating agents that modify the O6-position of guanine. Thus, inhibition of MGMT activity in tumors has a great interest for cancer researchers because it can significantly improve the anticancer efficacy of such alkylating agents. In this study, we performed a quantitative structure activity relationship (QSAR) and classification study based on a total of 134 base analogs related to their ED50 values (50% inhibitory concentration) against MGMT. Molecular information of all compounds were described by quantum chemical descriptors and Dragon descriptors. Genetic algorithm (GA) and multiple linear regression (MLR) analysis were combined to develop QSAR models. Classification models were generated by seven machine-learning methods based on six types of molecular fingerprints. Performances of all developed models were assessed by internal and external validation techniques. The best QSAR model was obtained with Q2Loo = 0.83, R2 = 0.87, Q2ext = 0.67, and R2ext = 0.69 based on 84 compounds. The results from QSAR studies indicated topological charge indices, polarizability, ionization potential (IP), and number of primary aromatic amines are main contributors for MGMT inhibition of base analogs. For classification studies, the accuracies of 10-fold cross-validation ranged from 0.750 to 0.885 for top ten models. The range of accuracy for the external test set ranged from 0.800 to 0.880 except for PubChem-Tree model, suggesting a satisfactory predictive ability. Three models (Ext-SVM, Ext-Tree and Graph-RF) showed high and reliable predictive accuracy for both training and external test sets. In addition, several representative substructures for characterizing MGMT inhibitors were identified by information gain and substructure frequency analysis method. Our studies might be useful for further study to design and rapidly identify potential MGMT inhibitors.

scholarly journals SDS2 CHALLENGES IN TREATMENT OF GLIOBLASTOMA: CURRENT CONCEPTS AND THERAPEUTIC PERSPECTIVES

2019 ◽  
Vol 1 (Supplement_2) ◽  
pp. ii3-ii3
Author(s):  
Wolfgang Wick
Keyword(s):  
Network Architecture ◽  
Dna Methyltransferase ◽  
Molecular Mechanisms ◽  
Treatment Resistance ◽  
Secondary Resistance ◽  
Methylguanine Dna Methyltransferase ◽  
Clinical Investigations ◽  
Mgmt Promoter ◽  
Brain Interface ◽  
First Time

Abstract The natural disease course in glioblastoma is grim, in adults as well as in children. To date, there are no options for primary, secondary or tertiary prevention. However, unlike the fatalistic approach generally taken, there are subgroups of patients or individuals clearly benefitting over a variable time from current treatments, radiation and alkylating chemotherapy, as well as experimental precision or immune interventions. This heterogeneity in treatment response reflects the biological heterogeneity of the disease, which needs to be addressed in current preclinical and clinical investigations as well as this identifies primary and acquired treatment resistance as the key challenge in the field of glioblastoma. Importantly, even for most conventional treatments the basic molecular mechanisms for primary or secondary resistance are unknown or incompletely understood. The present view is that progress will be made with a more precise classification and grouping of glioblastoma. The methylation subgroups clearly provide a first step, but further tumor bulk but potentially also subclonal or single-cell analyses might provide further insights and will be a prerequisite to meaningfully interpretable trials. Novel preclinical and translational concepts of glioblastoma in adults reflecting the proposed network architecture of the glioma, but also the glioma-brain interface may for the first time separate options for trial interventions in glioblastoma form the usual mainstream in oncology. Clinical trials of the past years have revealed the potential for further developing a lomustine/temozolomide combination in O6-methylguanine DNA-methyltransferase (MGMT) promoter hypermethylated glioblastoma and allow leaving out temozolomide for glioblastoma harboring an unmethylated MGMT promoter. The latter is not clinical standard, however we should at some point make sure we still understand, why temozolomide is provided in this biological situation and how we make a next step. The field of immunoneurooncology is rapidly growing with preclinical work and trial concepts, but whereas patients with brain metastases seem to benefit from this development, success in glioblastoma is restricted to uncontrolled earl-phase developments.

Specific Recognition ofO6-Methylguanine in DNA by Active Site Mutants of HumanO6-Methylguanine-DNA Methyltransferase†

Biochemistry ◽  
1997 ◽  
Vol 36 (19) ◽  
pp. 5769-5776 ◽  
Author(s):  
Tapas K. Hazra ◽  
Rabindra Roy ◽  
Tapan Biswas ◽  
David T. Grabowski ◽  
Anthony E. Pegg ◽  
...  
Keyword(s):  
Active Site ◽  
Dna Methyltransferase ◽  
Methylguanine Dna Methyltransferase ◽  
Specific Recognition ◽  
Active Site Mutants

scholarly journals Crystal structures of two monomeric triosephosphate isomerase variants identifiedviaa directed-evolution protocol selecting forL-arabinose isomerase activity

2016 ◽  
Vol 72 (6) ◽  
pp. 490-499
Author(s):  
Mirja Krause ◽  
Tiila-Riikka Kiema ◽  
Peter Neubauer ◽  
Rik K. Wierenga
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Directed Evolution ◽  
Crystal Structures ◽  
Active Site ◽  
Triosephosphate Isomerase ◽  
Point Mutations ◽  
Van Der Waals Interactions ◽  
Side Chain ◽  
Arabinose Isomerase

The crystal structures are described of two variants of A-TIM: Ma18 (2.7 Å resolution) and Ma21 (1.55 Å resolution). A-TIM is a monomeric loop-deletion variant of triosephosphate isomerase (TIM) which has lost the TIM catalytic properties. Ma18 and Ma21 were identified after extensive directed-evolution selection experiments using anEscherichia coliL-arabinose isomerase knockout strain expressing a randomly mutated A-TIM gene. These variants facilitate better growth of theEscherichia coliselection strain in medium supplemented with 40 mML-arabinose. Ma18 and Ma21 differ from A-TIM by four and one point mutations, respectively. Ma18 and Ma21 are more stable proteins than A-TIM, as judged from CD melting experiments. Like A-TIM, both proteins are monomeric in solution. In the Ma18 crystal structure loop 6 is open and in the Ma21 crystal structure loop 6 is closed, being stabilized by a bound glycolate molecule. The crystal structures show only small differences in the active site compared with A-TIM. In the case of Ma21 it is observed that the point mutation (Q65L) contributes to small structural rearrangements near Asn11 of loop 1, which correlate with different ligand-binding properties such as a loss of citrate binding in the active site. The Ma21 structure also shows that its Leu65 side chain is involved in van der Waals interactions with neighbouring hydrophobic side-chain moieties, correlating with its increased stability. The experimental data suggest that the increased stability and solubility properties of Ma21 and Ma18 compared with A-TIM cause better growth of the selection strain when coexpressing Ma21 and Ma18 instead of A-TIM.

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