RESUMEN
Thermus thermophilus is an extremely thermophilic organism that thrives at a temperature of 65°C. T. thermophilus genome has ~2218 genes, out of which 66% (1482 genes) have been annotated, and the remaining 34% (736 genes) are assigned as hypothetical proteins. In this work, biochemical and biophysical experiments were performed to characterize the hypothetical protein TTHA1873 from T. thermophilus. The hypothetical protein TTHA1873 acts as a nuclease, which indiscreetly cuts methylated and non-methylated DNA in divalent metal ions and relaxes the plasmid DNA in the presence of ATP. The chelation of metal ions with EDTA inhibits its activity. These results suggest that the hypothetical protein TTHA1873 would be a CRISPR-associated protein with non-specific DNase activity and ATP-dependent DNA-relaxing activity.
Asunto(s)
Proteínas Bacterianas , Thermus thermophilus , Thermus thermophilus/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Plásmidos/genética , Temperatura , Adenosina Trifosfato/metabolismoRESUMEN
RecFOR pathway is the principal repair pathway for double strand break and single strand gap repair in Thermus thermophilus. RecF and RecR exist as monomer and dimer in solution, interestingly; they undergo condition-dependent dimerization and tetramerization, respectively during the DNA break repair. However, their importance in protein-protein and protein-DNA interactions remains elusive. In this study, the three-dimensional crystal structures of the wild type RecF and RecR proteins are determined. Thereafter, the structural information is used to mutate the interface residues to cysteine to stabilize the dimeric and tetrameric states of the RecF and RecR proteins, respectively. A comparative study for their interactions with other cognate proteins and ssDNA in native and SSB (single strand binding protein) bound states was performed. RecF or RecFR complex displays a negligible affinity towards ssDNA. Conversely, the RecF mutants and its complexes with wild type RecR showed affinity towards ssDNA, suggesting, distinct modes of interaction of RecF and RecFR complex for ssDNA binding. In the presence of RecO, the stabilized tetrameric RecR showed a lower binding affinity for ssDNA as compared to the SSB bound ssDNA, indicating the importance of tetrameric RecR in stabilizing the RecOR complex on the SSB coated ssDNA. This provides an insight into the reduction of the binding affinity of SSB proteins with the ssDNA, which in turn enhances the recruitment of RecA for strand exchange.
Asunto(s)
Proteínas Bacterianas/química , Proteínas de Unión al ADN/química , Multimerización de Proteína , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Reparación del ADN , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica , Transducción de Señal , Relación Estructura-Actividad , Thermus thermophilusRESUMEN
Several studies on enzyme catalysis have pointed out that the product release event could be a rate limiting step. In this study, we have compared the release event of two products, Adenosine di-phosphate (ADP) and Thymidine di-phosphate (TDP) from the active-site of human and Thermus thermophilus thymidine mono-phosphate kinase (TMPK), referred to as hTMPK and ttTMPK, respectively. TMPK catalyses the conversion of Thymidine mono-phosphate (TMP) to TDP using ATP as phosphoryl donor in the presence of Mg2+ ion. Most of the earlier studies on this enzyme have focused on understanding substrate binding and catalysis, but the critical product release event remains elusive. Competitive binding experiments of the substrates and the products using ttTMPK apo crystals have indicated that the substrate (TMP) can replace the bound product (TDP), even in the presence of an ADP molecule. Further, the existing random accelerated molecular dynamics (RAMD) simulation program was modified to study the release of both the products simultaneously from the active site. The RAMD simulations on product-bound structures of both ttTMPK and hTMPK, revealed that while several exit patterns of the products are permissible, the sequential exit mode is the most preferred pattern for both ttTMPK and hTMPK enzymes. Additionally, the product release from the hTMPK was found to be faster and more directional as compared to ttTMPK. Structural investigation revealed that the critical changes in the residue composition in the LID-region of ttTMPK and hTMPK have an effect on the product release and can be attributed to the observed differences during product release event. Understanding of these dissimilarities is of considerable utility in designing potent inhibitors or prodrugs that can distinguish between eukaryotic and prokaryotic homologues of thymidylate kinase.
