@article {30082, title = {Structural basis for substrate discrimination by E. coli repair enzyme, AlkB}, journal = {RSC ADVANCES}, volume = {8}, year = {2018}, month = {2018}, pages = {1281-1291}, abstract = {

E. coli AlkB, a repair enzyme of the dioxygenase family, catalyses the removal of mutagenic methylated nucleotides from the genome. Known for substrate promiscuity, AlkB{\textquoteright}s catalytic mechanism and conformational changes accompanying substrate binding have been extensively dissected. However, the structural parameters of various substrates governing their recognition by AlkB still remain elusive. In this work, through solution-state vibrational spectra of methylated substrates bound to AlkB in combination with computational analysis, we show that the recognition specificity is dictated by the protonation states of the substrates. Specificity is conferred predominantly through hydrogen bonding and cation{\textendash}π interactions. Furthermore, we report on the interaction of AlkB with normal, unmodified nucleotides, wherein the presence of an exocyclic amino group serves as an essential criterion for the initial process of substrate recognition. Taken together, these results provide a rationale for structural determinants of substrate specificity as well as mode of lesion discrimination employed by AlkB.

}, doi = {10.1039/c7ra11333a}, author = {Jayanth, Namrata and Ogirala, Nirmala and Yadav, Anil and Puranik, Mrinalini} } @article {18680, title = {Mechanism of Discrimination of 8-Oxoguanosine versus Guanosine by Escherichia coli Fpg.}, journal = {J Phys Chem B}, year = {2017}, month = {2017 Jun 07}, abstract = {

The mutagenic 8-oxoguanosine monophosphate, the predominant product of DNA oxidation, is excised by formamidopyrimidine glycosylase (Fpg) in bacteria. The mechanism of recognition of 8-oxodG, which differs subtly from its normal counterpart, guanosine monophosphate (dG), by Escherichia coli Fpg remains elusive due to the lack of structural data of E. coli Fpg bound to 8-oxodG. Here, we present solution-state structure of 8-oxodG oligomer bound to E. coli E3Q Fpg using UV resonance Raman (UVRR) spectroscopy. The vibrational spectra report on the π-stacking and hydrogen bonding interactions established by 8-oxodG with E. coli E3Q Fpg. Furthermore, we report on the interactions of E. coli E3Q Fpg with the normal, undamaged nucleotide, dG. We show that E. coli Fpg recognizes 8-oxodG and dG through their C2-amino group but only 8-oxodG forms extensive contacts with E. coli Fpg. Our findings provide a basis for mechanism of lesion recognition by E. coli Fpg.

}, issn = {1520-5207}, doi = {10.1021/acs.jpcb.7b00205}, author = {Jayanth, Namrata and Puranik, Mrinalini} } @article {1015, title = {Differential Distortion of Purine Substrates by Human and Plasmodium falciparum Hypoxanthine-Guanine Phosphoribosyltransferase to Catalyse the Formation of Mononucleotides.}, journal = {Chemphyschem}, volume = {16}, year = {2015}, month = {2015 Jul 20}, pages = {2172-81}, abstract = {

Plasmodium falciparum (Pf) hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a potential therapeutic target. Compared to structurally homologous human enzymes, it has expanded substrate specificity. In this study, 9-deazapurines are used as in situ probes of the active sites of human and Pf HGPRTs. Through the use of these probes it is found that non-covalent interactions stabilise the pre-transition state of the HGPRT-catalysed reaction. Vibrational spectra reveal that the bound substrates are extensively distorted, the carbonyl bond of nucleobase moiety is weakened and the substrate is destabilised along the reaction coordinate. Raman shifts of the human and Pf enzymes are used to quantify the differing degrees of hydrogen bonding in the homologues. A decreased Raman cross-section in enzyme-bound 9-deazaguanine (9DAG) shows that the phenylalanine residue (Phe186 in human and Phe197 in Pf) of HGPRT stacks with the nucleobase. Differential loss of the Raman cross-section suggests that the active site is more compact in human HGPRT as compared to the Pf enzyme, and is more so in the phosphoribosyl pyrophosphate (PRPP) complex 9DAG-PRPP-HGPRT than in 9-deazahypoxanthine (9DAH)-PRPP-HGPRT.

}, issn = {1439-7641}, doi = {10.1002/cphc.201500084}, author = {Karnawat, Vishakha and Gogia, Spriha and Balaram, Hemalatha and Puranik, Mrinalini} } @article {878, title = {Formation of a CH-π contact in the core of native barstar during folding.}, journal = {J Phys Chem B}, volume = {119}, year = {2015}, month = {2015 Feb 19}, pages = {2928-32}, abstract = {

An important part of the protein folding process is the consolidation of the protein core through the formation of specific, directional contacts after the initial hydrophobic collapse. Here, we simultaneously monitor formation of core contacts and assembly of secondary structure through salt-induced folding by using resonance Raman spectroscopy. Unfolded barstar at pH 12 was refolded by gradual addition of sodium sulfate salt. Altered spectral characteristics of the Trp53 residue suggest that the core of the protein attains a CH-π interaction at a low concentration of the salt, with an increase in the packing density. Further increase in salt concentration produces a reduction in the solvent accessibility of the core. These data provide evidence that the core of the protein becomes rigid upon the addition of 0.6 M sodium sulfate. This is the first time that the formation of a CH-π interaction has been directly monitored during the folding of a protein.

}, issn = {1520-5207}, doi = {10.1021/jp512036p}, author = {Mil{\'a}n-Garc{\'e}s, Erix A and Thaore, Pallavi and Udgaonkar, Jayant B and Puranik, Mrinalini} }