Biochemical and Structural Studies of the Mycobacterium tuberculosis
            <i>O</i>
            <sup>6</sup>
            -Methylguanine Methyltransferase and Mutated Variants

Miggiano, Riccardo; Casazza, V.; Garavaglia, Silvia; Ciaramella, Maria; Perugino, Giuseppe; Rizzi, Menico; Rossi, Franca

doi:10.1128/jb.02298-12

Cited by 29 publications

(45 citation statements)

References 48 publications

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“…Despite the different primary structures (Figure 2a), thermophilic enzymes show a typical AGT protein architecture, consisting of two domains [37]: a highly conserved C-terminal domain (CTD), surprisingly superimposable for all available AGT structures (Figure 2b), and a N-terminal domain (NTD), which is very different among AGTs and whose function is not well understood (likely involved in regulation, cooperative binding, and stability [6,38,39]). The CTD contains the DNA binding helix-turn-helix motif (HTH); the Asn hinge, which precedes the -V/IPCHRVV/I-amino acid sequence containing the conserved catalytic cysteine (except the Caenorhabditis elegans AGT-2 that has the -PCHPsequence [40,41]); and the active site loop, responsible for the substrate specificity.…”

Section: The Common Themes In Agts' Tertiary Structure and The Intrismentioning

confidence: 99%

“…As described in Table 2, SsOGT shows the smaller total SASA value, in line with its exceptional stability. On the contrary, OGT from Mycobacterium tuberculosis [38,46] has a higher value due to the peculiar conformation of both the active site loop and the C-terminal tail that are exposed to the bulk solvent and are less heat stable (Table 2).…”

Section: The Common Themes In Agts' Tertiary Structure and The Intrismentioning

confidence: 99%

“…Because AGT covalently binds a benzyl-fluorescein moiety of its substrate after reaction, it is possible to immediately load the protein product on a SDS-PAGE-the gel-imaging analysis of the fluorescence intensity gives a direct measure of the protein activity because of the 1:1 stoichiometry of protein/substrate (Figure 3). Signals of fluorescent protein (corrected by the amount of loaded protein by Coomassie staining analysis) obtained at different times are plotted, and a second order reaction rate is determined [38,39,46,52,59,60]. This method can be applied to all AGTs that bind O 6 -BG, with the exception of the E. coli Ada-C [61,62].…”

Section: Innovative Ogt Assaysmentioning

confidence: 99%

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O6-alkylguanine-DNA Alkyltransferases in Microbes Living on the Edge: From Stability to Applicability

Mattossovich

Merlo

Miggiano

et al. 2020

IJMS

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The genome of living cells is continuously exposed to endogenous and exogenous attacks, and this is particularly amplified at high temperatures. Alkylating agents cause DNA damage, leading to mutations and cell death; for this reason, they also play a central role in chemotherapy treatments. A class of enzymes known as AGTs (alkylguanine-DNA-alkyltransferases) protects the DNA from mutations caused by alkylating agents, in particular in the recognition and repair of alkylated guanines in O6-position. The peculiar irreversible self-alkylation reaction of these enzymes triggered numerous studies, especially on the human homologue, in order to identify effective inhibitors in the fight against cancer. In modern biotechnology, engineered variants of AGTs are developed to be used as protein tags for the attachment of chemical ligands. In the last decade, research on AGTs from (hyper)thermophilic sources proved useful as a model system to clarify numerous phenomena, also common for mesophilic enzymes. This review traces recent progress in this class of thermozymes, emphasizing their usefulness in basic research and their consequent advantages for in vivo and in vitro biotechnological applications.

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Section: The Common Themes In Agts' Tertiary Structure and The Intrismentioning

confidence: 99%

Section: The Common Themes In Agts' Tertiary Structure and The Intrismentioning

confidence: 99%

Section: Innovative Ogt Assaysmentioning

confidence: 99%

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O6-alkylguanine-DNA Alkyltransferases in Microbes Living on the Edge: From Stability to Applicability

Mattossovich

Merlo

Miggiano

et al. 2020

IJMS

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“…In particular, MTB shows a gene fusion of adaA with alkA (AdaA-AlkA), and an independent adaB gene, also annotated as ogt, which encodes for the OGT protein (Rv1316c). MTB OGT, as the orthologous proteins of other organisms, invariably performs the removal of alkyl adducts on modified guanines through a suicidal mechanism, by catalyzing the stoichiometric transfer of the O6-alkyl group to the strictly conserved cysteine residue in the protein active site, which is hosted in the C-terminal domain [93][94][95]. The protein's overall structure and mechanistic aspects of the suicidal reaction are highly conserved among prokaryotic OGTs, as well as in the human equivalent enzyme [96][97][98][99][100], which is a validated target for cancer chemotherapy [101].…”

Section: Targeting Mtb Dna Repairmentioning

confidence: 99%

Targeting Genome Integrity in Mycobacterium Tuberculosis: From Nucleotide Synthesis to DNA Replication and Repair

et al. 2020

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Mycobacterium tuberculosis (MTB) is the causative agent of tuberculosis (TB), an ancient disease which still today causes 1.4 million deaths worldwide per year. Long-term, multi-agent anti-tubercular regimens can lead to the anticipated non-compliance of the patient and increased drug toxicity, which in turn can contribute to the emergence of drug-resistant MTB strains that are not susceptible to first- and second-line available drugs. Hence, there is an urgent need for innovative antitubercular drugs and vaccines. A number of biochemical processes are required to maintain the correct homeostasis of DNA metabolism in all organisms. Here we focused on reviewing our current knowledge and understanding of biochemical and structural aspects of relevance for drug discovery, for some such processes in MTB, and particularly DNA synthesis, synthesis of its nucleotide precursors, and processes that guarantee DNA integrity and genome stability. Overall, the area of drug discovery in DNA metabolism appears very much alive, rich of investigations and promising with respect to new antitubercular drug candidates. However, the complexity of molecular events that occur in DNA metabolic processes requires an accurate characterization of mechanistic details in order to avoid major flaws, and therefore the failure, of drug discovery approaches targeting genome integrity.

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“…Los análisis en conjunto de cepas MDR y XDR determinan qué metiltransferasas se encuentran con más variabilidad, y esto debido probablemente a su rol en la reparación del ADN de MTB lo que provee una estabilidad genética, a pesar de la exposición del entorno hostil de macrófagos (28) , además de actuar como activador de drogas, por ejemplo a tiacetazona (29) . También el hecho de que genes involucrados en la síntesis de riboflavina, cobalamina, glutamina, molibdeno, nicotidamina y magnesio chelatasa se encuentren exclusivamente en las MDR y XDR, lleva a considerar que MTB y su cambio de fitness (eficacia biológica) presionan que existan modificaciones o selección dentro de las drogorresistentes para sintetizar moléculas clave en su funcionamiento normal o la modificación de alternativas en sus rutas metabólicas (30) .…”

Section: Ins-benunclassified

Análisis genómico comparativo de cepas peruanas de Mycobacterium tuberculosis

Tarazona

Galarza

Levano

et al. 2016

Rev Peru Med Exp Salud Publica

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Objetivos. Analizar comparativamente tres secuencias genómicas de Mycobacterium tuberculosis (MTB): INS-SEN, cepa sensible; INS-MDR, cepa multidrogorresistente e INS-XDR, cepa extensamente resistente, procedentes de la Ciudad de Lima, Perú. Materiales y métodos. Se identificaron los polimorfismos de un solo nucleótido (SNPs) específicos en las cepas INS-SEN, INS-MDR y INS-XDR mediante el criterio de inclusión/exclusión. Se compararon los tres genomas de MTB y se construyó una filogenia molecular con 27 cepas de MTB de otros estudios, disponibles de la base de datos Genbank. Los SNPs específicos en cada genoma fueron organizados en clústers de grupos ortólogos (COGs). Resultados. El análisis de genomas permitió identificar un conjunto de SNPs asociados a determinantes de virulencia (familia de proteínas mce, policetidos, phiRv1, transposasas, metiltransferasas y relacionados a síntesis de vitaminas) principalmente. Se observa una estrecha relación entre la cepa INS-MDR y INS-XDR, con solo un 6,1% de SNPs diferentes, sin embargo, la cepa INS-SEN presenta un 50,2 y 50,3% de SNPs diferentes a las cepas MDR y XDR, respectivamente. La filogenia molecular agrupó a las cepas peruanas dentro del linaje LAM y cercanamente a las cepas F11 y KZN de Sudáfrica. Conclusiones. Se evidenció una alta similitud (99,9%) de la cepa INS-SEN con la cepa sudafricana F11, de gran alcance mundial, mientras los análisis de las cepas INS-MDR e INS-XDR demuestran una probable expansión de la familia KZN, cepa de Sudáfrica con alta virulencia y patogenicidad.Palabras clave: Tuberculosis; Genómica; Farmacorresistencia bacteriana (fuente: DeCS BIREME). COMPARATIVE GENOMIC ANALYSIS OF PERUVIAN STRAINS OF Mycobacterium tuberculosis ABSTRACT Objectives. To comparatively analyze three genomic sequences of Mycobacterium tuberculosis (MTB), including sensitive (INS-SEN), multi-drug-resistant (INS-MDR), and extremely drug-resistant (INS-XDR) strains, collected in Lima,Peru. Materials and Methods. Specific single nucleotide polymorphisms (SNPs) were identified in the INS SEN, INS-MDR, and INS-XDR strains according to the inclusion/exclusion criteria. The three MTB genomes were compared and a molecular phylogeny was constructed with 27 MTB strains from other studies available from the Genbank database. Results. The specific SNPs in each genome were organized in clusters of orthologous groups (COGs). The genomic analysis allowed for the identification of a set of SNPs associated mainly with virulence determinants (family of mce proteins, polyketides, phiRv1, transposase, and methyltransferases, and other related to vitamin synthesis). A close correlation between the INS-MDR and INS-XDR strains was observed, with only a 6.1% difference in SNPs; however, the INS-SEN strain had 50.2% and 50.3% different SNPs from the MDR and XDR strains, respectively. The molecular phylogeny grouped the Peruvian strains within the LAM lineage and closely to the F11 and KZN strains from South Africa. Conclusions. High similarity (99.9%) was noted between the INS-SEN strain ...

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Biochemical and Structural Studies of the Mycobacterium tuberculosis O ⁶ -Methylguanine Methyltransferase and Mutated Variants

Cited by 29 publications

References 48 publications

O6-alkylguanine-DNA Alkyltransferases in Microbes Living on the Edge: From Stability to Applicability

O6-alkylguanine-DNA Alkyltransferases in Microbes Living on the Edge: From Stability to Applicability

Targeting Genome Integrity in Mycobacterium Tuberculosis: From Nucleotide Synthesis to DNA Replication and Repair

Análisis genómico comparativo de cepas peruanas de Mycobacterium tuberculosis

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Biochemical and Structural Studies of the Mycobacterium tuberculosis O 6 -Methylguanine Methyltransferase and Mutated Variants

Cited by 29 publications

References 48 publications

O6-alkylguanine-DNA Alkyltransferases in Microbes Living on the Edge: From Stability to Applicability

O6-alkylguanine-DNA Alkyltransferases in Microbes Living on the Edge: From Stability to Applicability

Targeting Genome Integrity in Mycobacterium Tuberculosis: From Nucleotide Synthesis to DNA Replication and Repair

Análisis genómico comparativo de cepas peruanas de Mycobacterium tuberculosis

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Biochemical and Structural Studies of the Mycobacterium tuberculosis O ⁶ -Methylguanine Methyltransferase and Mutated Variants