Functional analysis of amino acid residues at the dimerisation interface of KpnI DNA methyltransferase

Bheemanaik, Shivakumara; Bujnicki, Janusz M.; Nagaraja, Valakunja; Rao, Desirazu N.

doi:10.1515/bc.2006.067

Cited by 14 publications

(15 citation statements)

References 47 publications

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“…The Y. pestis enzyme shows a considerably lower K M DNA than that reported for the E. coli protein [17]. The observed k cat for Y. pestis Dam is 0.55±0.01 min −1 , a similar rate to that reported for both the E. coli and T4 DNA adenine methyltransferases [31]. The error in K M AdoMet and k cat were comparable to those obtained using the tritium labelled filter binding assay.…”

Section: Resultssupporting

confidence: 81%

Kinetic Analysis of Yersinia pestis DNA Adenine Methyltransferase Activity Using a Hemimethylated Molecular Break Light Oligonucleotide

et al. 2007

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BackgroundDNA adenine methylation plays an important role in several critical bacterial processes including mismatch repair, the timing of DNA replication and the transcriptional control of gene expression. The dependence of bacterial virulence on DNA adenine methyltransferase (Dam) has led to the proposal that selective Dam inhibitors might function as broad spectrum antibiotics.Methodology/Principal FindingsHerein we report the expression and purification of Yersinia pestis Dam and the development of a continuous fluorescence based assay for DNA adenine methyltransferase activity that is suitable for determining the kinetic parameters of the enzyme and for high throughput screening against potential Dam inhibitors. The assay utilised a hemimethylated break light oligonucleotide substrate containing a GATC methylation site. When this substrate was fully methylated by Dam, it became a substrate for the restriction enzyme DpnI, resulting in separation of fluorophore (fluorescein) and quencher (dabcyl) and therefore an increase in fluorescence. The assays were monitored in real time using a fluorescence microplate reader in 96 well format and were used for the kinetic characterisation of Yersinia pestis Dam, its substrates and the known Dam inhibitor, S-adenosylhomocysteine. The assay has been validated for high throughput screening, giving a Z-factor of 0.71±0.07 indicating that it is a sensitive assay for the identification of inhibitors.Conclusions/SignificanceThe assay is therefore suitable for high throughput screening for inhibitors of DNA adenine methyltransferases and the kinetic characterisation of the inhibition.

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Section: Resultssupporting

confidence: 81%

Kinetic Analysis of Yersinia pestis DNA Adenine Methyltransferase Activity Using a Hemimethylated Molecular Break Light Oligonucleotide

et al. 2007

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“…Circular permutations have been reported in various DNA and RNA MTases from the RFM superfamily [72-74]. It has been speculated that permutation could have played a role in the evolution of new specificities and appearance of dimerization in DNA MTases [75,76]. In the SPOUT domain the N- and C-termini are adjacent in space, suggesting that the C-terminal helix could be covalently linked to the N-terminal strand without the need of a long linker.…”

Section: Resultsmentioning

confidence: 99%

Structural and evolutionary bioinformatics of the SPOUT superfamily of methyltransferases

et al. 2007

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Background: SPOUT methyltransferases (MTases) are a large class of S-adenosyl-L-methionine-dependent enzymes that exhibit an unusual alpha/beta fold with a very deep topological knot. In 2001, when no crystal structures were available for any of these proteins, Anantharaman, Koonin, and Aravind identified homology between SpoU and TrmD MTases and defined the SPOUT superfamily. Since then, multiple crystal structures of knotted MTases have been solved and numerous new homologous sequences appeared in the databases. However, no comprehensive comparative analysis of these proteins has been carried out to classify them based on structural and evolutionary criteria and to guide functional predictions.

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“…In addition, β-class methyltransferases such as LlaCI MTase [39] and KpnI MTase [40] have been shown to dimerize in solution. In case of KpnI methyltransferase dimerization has been shown to be required for high affinity substrate binding needed for catalysis [41].…”

Section: Resultsmentioning

confidence: 99%

Functional Analysis of an Acid Adaptive DNA Adenine Methyltransferase from Helicobacter pylori 26695

Banerjee

Rao

2011

PLoS ONE

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HP0593 DNA-(N6-adenine)-methyltransferase (HP0593 MTase) is a member of a Type III restriction-modification system in Helicobacter pylori strain 26695. HP0593 MTase has been cloned, overexpressed and purified heterologously in Escherichia coli. The recognition sequence of the purified MTase was determined as 5′-GCAG-3′and the site of methylation was found to be adenine. The activity of HP0593 MTase was found to be optimal at pH 5.5. This is a unique property in context of natural adaptation of H. pylori in its acidic niche. Dot-blot assay using antibodies that react specifically with DNA containing m6A modification confirmed that HP0593 MTase is an adenine-specific MTase. HP0593 MTase occurred as both monomer and dimer in solution as determined by gel-filtration chromatography and chemical-crosslinking studies. The nonlinear dependence of methylation activity on enzyme concentration indicated that more than one molecule of enzyme was required for its activity. Analysis of initial velocity with AdoMet as a substrate showed that two molecules of AdoMet bind to HP0593 MTase, which is the first example in case of Type III MTases. Interestingly, metal ion cofactors such as Co2+, Mn2+, and also Mg2+ stimulated the HP0593 MTase activity. Preincubation and isotope partitioning analyses clearly indicated that HP0593 MTase-DNA complex is catalytically competent, and suggested that DNA binds to the MTase first followed by AdoMet. HP0593 MTase shows a distributive mechanism of methylation on DNA having more than one recognition site. Considering the occurrence of GCAG sequence in the potential promoter regions of physiologically important genes in H. pylori, our results provide impetus for exploring the role of this DNA MTase in the cellular processes of H. pylori.

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Functional analysis of amino acid residues at the dimerisation interface of KpnI DNA methyltransferase

Cited by 14 publications

References 47 publications

Kinetic Analysis of Yersinia pestis DNA Adenine Methyltransferase Activity Using a Hemimethylated Molecular Break Light Oligonucleotide

Kinetic Analysis of Yersinia pestis DNA Adenine Methyltransferase Activity Using a Hemimethylated Molecular Break Light Oligonucleotide

Structural and evolutionary bioinformatics of the SPOUT superfamily of methyltransferases

Functional Analysis of an Acid Adaptive DNA Adenine Methyltransferase from Helicobacter pylori 26695

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