BamHI
From Proteopedia
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is a type II restriction enzyme derived from Bacillus amyloliquefaciens. It recognizes the DNA sequence of G’GATCC and leaves an overhang of GATC which is compatible with many other enzymes.[1] The BamHI-DNA complex is a sequence-specific endonucleases-DNA complex. In the complex every potential hydrogen bond donor and acceptor in the major groove of the recognition site takes part in direct or water-mediated hydrogen bonds with the protein. No other DNA sequence could support this degree of complementarity with BamHI.[2] There are five crystal structures of BamHI in the Protein Data Bank. These include BamHI bound to a non-specific DNA, BamHI complex with DNA and calcium ions (pre-reactive complex), BamHI complex with DNA and manganese ions (post-reactive complex), BamHI complex with DNA, and BamHI phased at 1.95 angstroms resolution by MAD analysis.[3]
Type II restriction enzymes require only Mg2+ as a cofactor to catalyze the hydrolysis of DNA phosphodiesters, leaving free 5’ phosphate and 3’ hydroxyl groups. The reaction is considered to proceed by an inline displacement of the 3’ leaving group, in which an activated water molecule acts as the attacking nucleophile. The active sites for BamHI are residues Asp94, Glu111, and Glu113. These can be spatially aligned with residues in EcoRI, EcoRV and PvuII. Several mechanisms have been proposed for the way these enzymes might activate a water molecule for nucleophilic attack. It is suggested there is a general base mechanism in which the acidic residues Asp94 and Glu111 coordinate Mg2+ at the active site while Glu113 acts as a general base to deprotonate the attacking water molecule. A separate mechanism has been suggested in which a water molecule is located centrally at the active site within hydrogen-bonding distances from the carboxylate groups of Glu111 and Glu113, the nonesterified oxygens of the phosphate group and the main chain carbonyl group of Phe112.[4]
The problem of specific versus nonspecific DNA selection is particularly acute for bacterial restriction enzymes. For every BamHI cognate DNA site in the Bacillus amyloliquefaciens H genome, there are 18 sites that differ by only a single base pair. Only the cognate sites are protected from cleavage by methylation produced by the partner methylase enzyme. The stringent specificity for a bacterial restriction enzyme can reduce their cleavage activity by a million-fold with a change in just one base pair. There is an exception in which BamHI binds to a noncognate DNA sequence GAATCC which differs by only one base pair from the cognate G’GATCC sequence. The structure reveals the enzyme is a distinct conformation that is incompetent for cleavage but competent for sliding. [5]
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