27 research outputs found
Endonuclease Active Site Plasticity Allows DNA Cleavage with Diverse Alkaline Earth and Transition Metal Ions
No full textA majority of enzymes show a high degree of specificity toward a particular metal ion in their catalytic reaction. However, Type II restriction endonuclease (REase) R.KpnI, which is the first member of the HNH superfamily of REases, exhibits extraordinary diversity in metal ion dependent DNA cleavage. Several alkaline earth and transition group metal ions induce high fidelity and promiscuous cleavage or inhibition depending upon their concentration. The metal ions having different ionic radii and co-ordination geometries readily replace each other from the enzyme's active site, revealing its plasticity. Ability of R KpnI to cleave DNA with both alkaline earth and transition group metal ions having varied ionic radii could imply utilization of different catalytic site(s). However, mutation of the invariant His residue of the HNH motif caused abolition of the enzyme activity with all of the cofactors, indicating that the enzyme follows a single metal ion catalytic mechanism for DNA cleavage. Indispensability of His in nucleophile activation together with broad cofactor tolerance of the enzyme indicates electrostatic stabilization function of metal ions during catalysis. Nevertheless, a second metal ion is recruited at higher concentrations to either induce promiscuity or inhibit the DNA cleavage. Regulation of the endonuclease activity and fidelity by a second metal ion binding is a unique feature of R.KpnI among REases and HNH nucleases. The active site plasticity of R.KpnI opens up avenues for redesigning cofactor specificities and generation of mutants specific to a particular metal ion
Evolution of sequence specificity in a restriction endonuclease by a point mutation
Get PDFRestriction endonucleases (REases) protect bacteria from invading foreign DNAs and are endowed with exquisite sequence specificity. REases have originated from the ancestral proteins and evolved new sequence specificities by genetic recombination, gene duplication, replication slippage, and transpositional events. They are also speculated to have evolved from nonspecific endonucleases, attaining a high degree of sequence specificity through point mutations. We describe here an example of generation of exquisitely site-specific REase from a highly-promiscuous one by a single point mutation
Ca<SUP>2+</SUP>-mediated site-specific DNA cleavage and suppression of promiscuous activity of KpnI restriction endonuclease
No full textThe characteristic feature of type II restriction endonucleases (REases) is their exquisite sequence specificity and obligate Mg2+ requirement for catalysis. Efficient cleavage of DNA only in the presence of Ca2+ ions, comparable with that of Mg2+, is previously not described. Most intriguingly, KpnI REase exhibits Ca2+-dependent specific DNA cleavage. Moreover, the enzyme is highly promiscuous in its cleavage pattern on plasmid DNAs in the presence of Mn2+ or Mg2+, with the complete suppression of promiscuous activity in the presence of Ca2+. KpnI methyltransferase does not exhibit promiscuous activity unlike its cognate REase. The REase binds to oligonucleotides containing canonical and mapped noncanonical sites with comparable affinities. However, the extent of cleavage is varied depending on the metal ion and the sequence. The ability of the enzyme to be promiscuous or specific may reflect an evolutionary design. Based on the results, we suggest that the enzyme KpnI represents an REase evolving to attain higher sequence specificity from an ancient nonspecific nuclease
Unveiling the Modulating Role of Extracellular pH in Permeation and Accumulation of Small Molecules in Subcellular Compartments of Gram-negative <i>Escherichia coli</i> using Nonlinear Spectroscopy
No full textQuantitative evaluation
of small molecule permeation and accumulation
in Gram-negative bacteria is important for drug development against
these bacteria. While these measurements are commonly performed at
physiological pH, Escherichia coli and many other
Enterobacteriaceae infect human gastrointestinal and urinary tracts,
where they encounter different pH conditions. To understand how external
pH affects permeation and accumulation of small molecules in E. coli cells, we apply second harmonic generation (SHG)
spectroscopy using SHG-active antimicrobial compound malachite green
as the probe molecule. Using SHG, we quantify periplasmic and cytoplasmic
accumulations separately in live E. coli cells, which
was never done before. Compartment-wise measurements reveal accumulation
of the probe molecule in cytoplasm at physiological and alkaline pH,
while entrapment in periplasm at weakly acidic pH and retention in
external solution at highly acidic pH. Behind such disparity in localizations,
up to 2 orders of magnitude reduction in permeability across the inner
membrane at weakly acidic pH and outer membrane at highly acidic pH
are found to play key roles. Our results unequivocally demonstrate
the control of external pH over entry and compartment-wise distribution
of small molecules in E. coli cells, which is a vital
information and should be taken into account in antibiotic screening
against E. coli and other Enterobacteriaceae members.
In addition, our results demonstrate the ability of malachite green
as an excellent SHG-indicator of changes of individual cell membrane
and periplasm properties of live E. coli cells in
response to external pH change from acidic to alkaline. This finding,
too, has great importance, as there is barely any other molecular
probe that can provide similar information
A new type II restriction endonuclease, OfoI from nonheterocystous cyanobacterium Oscillatoria foreaui
No full textAlthough restriction enzymes are widely distributed in nature, many bacterial genera are yet to be explored for the presence of this important class of enzymes. We have purified and characterized a new type II restriction endonuclease, OfoI from a nonheterocyst cyanobacterium Oscillatoria foreaui. The recognition sequence has been determined by primer extension analysis. The purified enzyme OfoI recognizes and cleaves the palindromic hexanucleotide 5'-C↓YCGRG-3', generating 5'-protruding ends
Dual Role for in Maintaining Structural Integrity and Inducing DNA Sequence Specificity in a Promiscuous Endonuclease
No full textWe describe two uncommon roles for in enzyme KpnI restriction endonuclease (REase). Among all of the REases studied, KpnI REase is unique in itsDNAbinding and cleavage characteristics. The enzyme is a poor discriminator of DNA
sequences, cleaving DNA in a promiscuous manner in the presence of . Unlike most Type II REases, the active site of the enzyme comprises an HNH motif, which can accommodate ,, or . Among these metal ions, and induce promiscuous cleavage by the enzyme, whereas -bound enzyme exhibits site-specific cleavage. Examination of the sequence of the protein revealed the presence of a zinc finger CCCH motif rarely found in proteins of prokaryotic origin. The zinc binding motif tightly coordinates zinc to provide a rigid structural framework for the enzyme needed for its function. In addition to this structural scaffold, another atom of zinc binds to the active site to induce high fidelity cleavage and suppress the - and -mediated promiscuous behavior of the enzyme. This is the first demonstration of distinct structural and catalytic roles for zinc in an enzyme, suggesting the distinct origin of KpnI REase
Endonuclease Active Site Plasticity Allows DNA Cleavage with Diverse Alkaline Earth and Transition Metal Ions
No full textA majority of enzymes show a high degree of specificity toward a particular metal ion in their catalytic reaction. However, Type II restriction endonuclease (REase) R.KpnI, which is the first member of the HNH superfamily of REases, exhibits extraordinary diversity in metal ion dependent DNA cleavage. Several alkaline earth and transition group metal ions induce high fidelity and promiscuous cleavage or inhibition depending upon their concentration. The metal ions having different ionic radii and co-ordination geometries readily replace each other from the enzyme’s active site, revealing its plasticity. Ability of R.KpnI to cleave DNA with both alkaline earth and transition group metal ions having varied ionic radii could imply utilization of different catalytic site(s). However, mutation of the invariant His residue of the HNH motif caused abolition of the enzyme activity with all of the cofactors, indicating that the enzyme follows a single metal ion catalytic mechanism for DNA cleavage. Indispensability of His in nucleophile activation together with broad cofactor tolerance of the enzyme indicates electrostatic stabilization function of metal ions during catalysis. Nevertheless, a second metal ion is recruited at higher concentrations to either induce promiscuity or inhibit the DNA cleavage. Regulation of the endonuclease activity and fidelity by a second metal ion binding is a unique feature of R.KpnI among REases and HNH nucleases. The active site plasticity of R.KpnI opens up avenues for redesigning cofactor specificities and generation of mutants specific to a particular metal ion
Structural integrity of the Beta Beta Alpha-Metal finger motif is required for DNA binding and stable protein-DNA complex formation in R.KpnI
No full textRestriction endonuclease (REase) R.KpnI from Klebsiella pneumoniae is a homodimeric enzyme, which recognizes palindromic sequence GGTAC|C and cleaves generating 4 base 3' end overhangs. R.KpnI belongs to the HNH superfamily of nucleases, which are characterized by the presence of the ββα-Me finger motif. Structurally, this motif consists of a twisted β-hairpin followed by an α-helix, and serves as a scaffold for side chains of residues involved in co-ordination of a divalent metal ion that is required for catalysis. Homology modeling studies of R.KpnI suggested a crossover structure for the α-helix, which could possibly form dimeric interface and/or structural scaffold for the active site. We have evaluated the role of the residues present in this α-helix in intersubunit interactions and/or stabilization of the active site. We show here that mutations of residues in the α-helix lead to a loss of the enzyme activity, but not dimerization ability. Intrinsic fluorescence and circular dichroism studies revealed that the loss of function phenotype was due to the structural perturbation of the ββα-Me finger motif. The results of mutational analysis suggest that the α-helix of the ββα-Me finger of R.KpnI plays an important role for the stability of the protein-DNA complex
Catechol dioxygenase is an insensitive reporter for transcription in Mycobacterium smegmatis
No full textAlthough restriction enzymes are widely distributed in nature, many bacterial genera are yet to be explored for the presence of this important class of enzymes. We have purified and characterized a new type II restriction endonuclease, OfoI from a nonheterocyst cyanobacterium Oscillatoria foreaui. The recognition sequence has been determined by primer extension analysis. The purified enzyme OfoI recognizes and cleaves the palindromic hexanucleotide 5¢-C¯YCGRG-3¢, generating 5¢-protruding ends
Exocyst subcomplex functions in autophagosome biogenesis by regulating Atg9 trafficking
No full textAbstractDuring autophagy, double membrane vesicles called autophagosomes capture and degrade the intracellular cargo. The de novo formation of autophagosomes requires several vesicle transport and membrane fusion events which are not completely understood. We studied the involvement of Exocyst- an octameric tethering complex, which has a primary function in tethering post-Golgi secretory vesicles to plasma membrane, in autophagy. Our findings indicate not all subunits of exocyst are involved in selective and general autophagy. We show that in the absence of autophagy specific subunits, autophagy arrest is accompanied by accumulation of incomplete autophagosome-like structures. In these mutants, impaired Atg9 trafficking leads to decreased delivery of membrane to the site of autophagosome biogenesis thereby impeding the elongation and completion of the autophagosomes. The subunits of exocyst which are dispensable for autophagic function do not associate with the autophagy specific subcomplex of exocyst.</jats:p
