Eco-RI endonuclease
Created by Andrew Vitek
Eco-RI endonuclease(1ERI) is a type II restriction enzyme found in Escherichia Coli. Type II restriction enzymes catalyze the hydrolysis of phosphodiester bonds found in specific DNA sequences. This facilitates DNA metabolism, which can act as a primitive defense against foreign DNA. Type II restriction enzymes are important in the fields of gene cloning and genetic engineering due to the specificity of restriction and the potential for recombination. Eco-RI endonuclease cleaves the specific hexaoligomeric sequence GAATTC at the guanine-adenine phosphodiester bond. The molecular weight of Eco-RI is 29,135.17 Da, and its isoelectric point (pI) is 6.23.
Eco-RI endonuclease exists as a homodimer where each monomer binds to either side of DNA(1). Eco-RI and other type II restriction endonucleases are enzymes that bind to specific oligonucleotide sequences and catalyze the hydrolysis of the phosphodiester bonds at specific that sequence without the use of ATP (2). Polar and charged residues face the DNA ligand in the binding site of the protein. As a homodimer, Eco-RI cleaves both sides of the nucleotide sequence GAATTC at the guanine-adenine phosphodiester bond. Lys-89 and Lys-113 bind to oligonucleotides adjacent to the guanine-adenine bond, which facilitates restriction in two ways. The amino side chains of Lys-89 and Lys-113 form hydrogen bonds with the phosphodiester backbone that facilitate both binding and electron density withdrawal. The adjacent adenine in the palindromic oligonucleotide sequence is methylated by methylase. When enough electron density is withdrawn, the phosphodiester bond is cleaved leaving a staggered cleavage where each of the fragments is cut to leave two overhanging nucleotide sequences, ATT (3). The specificity of restriction and the ability of the resulting fragments to be coordinated with other nucleotide sequences makes Type II restriction enzymes (e.g. Eco-RI) desirable topics of study (1). Gene cloning and other fields of genetic engineering depend on restriction enzymes for reconfiguring nucleic acid sequences. These proteins allow for the deletion or addition of nucleotide sequences that may express undesirable or desirable traits. Eco-RI has been shown to restrict sequences that differ slightly from the normal sequence in solutions of high pH or high ionic concentration, which provides further use of the protein in genetic engineering (4).
The mechanism by which Eco-RI cleaves DNA 5'-GAATTC-3' between guanine and adenine is an inversion of the phosphodiester backbone. Eco-RI forms a symmetric homodimer that binds at to the substrate. The enzyme-substrate complex has a kinked phosphodiester backbone at the scissile bond of guanine and adenine. This is the result of basepair interactions between adenine of the scissile bond and thymine of the complementary strand. Asn-141 and Arg-145 have bidentate hydrogen bonds with the DNA substrate at the scissile bond along the DNA backbone. Eco-RI has one catalytic site but binds to both strands of the double stranded DNA substrate in the homodimer of the enyzme-substrate complex. This allows for recognition of the palindromic DNA site, stabilization of the enzyme-substrate complex, and stabilization of the scissile bond in the binding site. When binding with Eco-RI, the major groove is widened by 0.35 nm, which allows for recognition of the substrate.
Substrate recognition is dependent on interactions with the enzyme and both strands of the double-stranded DNA substrate. Tyr-192 and Glu-193 are part of the protein chain that extends from the binding site on the primary strand of the DNA substrate to the complementary strand (not depicted). These residues lie between the active sites of the Eco-RI homodimer. This section of the enzyme is a random coil that traverses from the bound strand of the substrate to the complementary strand that is bound to the other subunit of the homodimer. Tyr-192 and Glu-193 recognize the thymine bases on the complementary strand. These interactions are thought to be part of the recognition model where substrate recognition and binding is dependent on interactions between the pyrimidine bases and Eco-RI.
The protein database search programs PSI-BLAST and the Dali database were used to find proteins with structures similar to Eco-RI. Both programs use algorithmic searches to find proteins of similar amino acid sequence and provide characteristic similarity scores. The purpose for searching for proteins of similar primary structure is to look for similarities between protein structure and biological function. The PSI-BLAST searches yielded several forms of the protein of interest crystallized with different ligands (5). The other proteins in the search had not been crystallized, so none of those proteins were used as comparisons with Eco-RI. The Dali Database search yielded a several proteins with significant similarities. Of these proteins, SgrAI was chosen as a protein with an intermediate Z-score, a characteristic number of the Dali Database that indicates the degree of structural similarity (6). The Expasy Bioinformatics database was used to acquire the molecular weight and isoelectric point of the protein. The functions of Eco-RI and SgrAI will be discussed and the structures of these proteins will be compared in order to inspect for functional similarities.
SgrAI is also a Type II restriction enzyme. At a superficial level, this is the first hint that structural similarity entails functional similarity. Both Eco-RI and SgrAI recognize and cut at specific sequences, but SgrAI is unique in that it cuts an 8-oligonucleotide sequence, CRCCGGYG (7). Both proteins recognize and cut at the same location of respective recognition sites. The length of Eco-RI is 276 amino acids while that of SgrAI is 338 (3,7). In order to cleave the two phosphodiester bonds of DNA, Eco-RI forms homodimers that bind on either side of the palindromic nucleotide sequence (7). The protein is known to have metal binding sites at the Asp-91 and Glu-111 sites. SgrAI is known to form a tetramer complexed with two Mn2+ or Ca2+ ions during DNA restriction (7). The secondary structures of the two proteins show small differences. The amino acid composition of Eco-RI is nearly 20% β-pleated sheets, less than 10% 3/10 helices, and over 50% α-helices. Nearly all of the α-helices appear on the exterior of the protein. SgrAI is composed of less than 10% β-pleated sheets, less than 10% 3/10 helices, and over 70% α-helices. Like Eco-RI, the majority of the α-helices of SgrAI appear on the protein exterior. Both proteins are globular in shape. Significant differences in protein function are found in DNA-substrate binding. Eco-RI binds to DNA through amino acids within 3/10 helical secondary structures. The metal cation facilitates the binding of SgrAI to the DNA substrate.
The primary structure of Eco-RI consists of 276 amino acids with a mean amino acid molecular weight of 113 Da. (8). This mean molecular weight does not deviate significantly from the average amino acid molecular weight of 110 Da., which implies that the protein does not have an irregular excess of large or small amino acids. The isoelectric point of the protein, which indicates the overall polarity of the protein, is 6.10 (8). The presence of several acidic amino acids is indicative of the types of bonds between the enzyme and substrate. Eco-RI binds to the phosphodiester backbone of DNA primarily via hydrogen bonds and polar interactions between the acidic side chains in the binding site of the enzyme and the negatively charged phosphate group of the phosphodiester backbone.
The secondary structure of Eco-RI shows a preponderance of α-helices and 3/10 helices. The α-helices are predominantly on the protein exterior while the two 3/10 helices reside in the protein interior. The residues of the 3/10 helices are composed of mostly polar side-chains. The interior portion of most globular proteins is generally composed of non-polar amino acids in order to maximize hydrophobic interactions. The presence of lengths of hydrophilic interior amino acids implies that the protein may be somewhat unstable or that these lengths of polar amino acids may be part of the active site.
The tertiary structure further elucidates the enzymatic activity of Eco-RI. The globular protein folds in such a way that allows most of the α-helices to be exposed to the cellular environment. The basic and polar residues of the protein’s interior random coils and β-sheets facilitate substrate binding. The amphipathic b-sheet illustrates common substrate-protein interactions. Polar and basic amino acids face the phosphodiester backbone of the ligand. These polar residues contribute to stronger interactions with the phosphodiester backbone of DNA, which facilitates substrate binding and bond scission. Specifically, basic amino acid side chains can participate in ionic and hydrogen bonds with the negatively charged phosphate groups of DNA. Nonpolar amino acids face the protein interior to maximize hydrophobic interactions and van der Waals interactions.