An Isoleucine to Leucine Mutation that Switches the Cofactor Requirement of the <i>Eco</i>RV Restriction Endonuclease from Magnesium to Manganese

Vipond, IB; Moon, Byung-Jo; Halford, Stephen E.

doi:10.1021/bi9523926

Cited by 47 publications

(40 citation statements)

References 32 publications

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“…However, unlike the restriction enzymes, PI-SceI normally displays greater activity in the presence of Mn 2ϩ than with Mg 2ϩ . One EcoRV mutant has been identified for which this is also the case (19). Taken together, our data are consistent with the Lys 301 side chain establishing an important binding contact within region I, perhaps to a phosphate oxygen near the scissile phosphodiester bond.…”

supporting

confidence: 84%

Amino Acid Residues in Both the Protein Splicing and Endonuclease Domains of the PI-SceI Intein Mediate DNA Binding

He¹,

Crist²,

Yen³

et al. 1998

Journal of Biological Chemistry

117

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A structure-based model describing the interaction of the two-domain PI-SceI endonuclease with its 31-base pair DNA substrate suggests that the endonuclease domain (domain II) contacts the cleavage site region of the substrate, while the protein splicing domain (domain I) interacts with a distal region that is sufficient for high affinity binding. To support this model, alanine-scanning mutagenesis was used to assemble a set of 49 PISceI mutant proteins that were purified and assayed for their DNA binding and cleavage properties. Fourteen mutant proteins were 4-to >500-fold less active than wild-type PI-SceI in cleavage assays, and one mutant (T225A) was 3-fold more active. The yeast PI-SceI endonuclease catalyzes the hydrolysis of two specific phosphodiester bonds within an asymmetrical recognition site (1). This enzyme is a homing endonuclease (for a review, see Ref. 2) that occurs as an intein situated within an H ϩ -ATPase protein subunit. Like other homing endonucleases, PI-SceI recognizes an extremely long sequence (31 bp) 1 and cuts DNA to yield 5Ј-phosphate and 3Ј-hydroxyl ends (3, 4). Mutagenesis and biochemical studies indicate that the PI-SceI recognition sequence can be divided into two regions (4, 5).Region I contains the cleavage site that is cut by the enzyme to generate a 4-base pair overhang, and region II includes an adjacent 17-bp sequence (the minimal binding sequence) that is sufficient for high affinity binding. Mutagenesis of the substrate reveals that PI-SceI tolerates substitutions at numerous positions, since substitutions at only nine positions in the substrate lead to severely reduced activity (4). Like the other homing endonucleases that have been studied, PI-SceI requires Mg 2ϩ as a cofactor. The metal ion is likely to be required for the hydrolytic reaction, since it is required for catalysis but not for specific binding. Mn 2ϩ can substitute for Mg 2ϩ , and it stimulates more efficient cleavage by the enzyme at cognate and noncognate sites (1, 5).The three-dimensional structure of PI-SceI has been recently determined by x-ray crystallography and reveals a bipartite domain structure (6). Domain I contains the protein splicing active site, which is composed of the N-and C-terminal amino acids and two other His residues that have been shown to be required for activity or have been implicated in the reaction (7,8). The residues that compose the putative endonucleolytic active site, a lysine (Lys 301 ) and two aspartic acid residues (Asp 218 and Asp 326 ), are present in domain II and form a catalytic triad that displays structural similarity to charged clusters found in restriction enzymes (6, 9). By using the PISceI structural information and the knowledge that the enzyme contacts two discrete regions of the recognition sequence, a model for the docking of PI-SceI with its substrate was constructed where domains I and II of the protein contact regions II and I, respectively, of the substrate (Fig. 1). In this model, both domains are proposed to contact the substrate, since the bindi...

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confidence: 84%

Amino Acid Residues in Both the Protein Splicing and Endonuclease Domains of the PI-SceI Intein Mediate DNA Binding

He¹,

Crist²,

Yen³

et al. 1998

Journal of Biological Chemistry

117

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“…Thus, manganese relaxes the topological specificity of PI-TfuI, but not its requirement for cognate sequence, which is the same with either metal on supercoiled DNA. In this respect, it is reminiscent of the I91L mutant of EcoRV, where a single mutation within the active site of the enzyme results in a total shift in metal requirement while retaining specificity for the cognate sequence (23). The longer sequence needed for cleavage of linear DNA could translate the requirement of initial DNA bending for efficient substrate binding.…”

Section: Discussionmentioning

confidence: 99%

Inteins of Thermococcus fumicolans DNA Polymerase Are Endonucleases with Distinct Enzymatic Behaviors

Saves¹,

Ozanne²,

Diétrich³

et al. 2000

Journal of Biological Chemistry

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The DNA polymerase gene of Thermococcus fumicolans harbors two intein genes. Both inteins have been produced in Escherichia coli and purified either as naturally spliced products from the expression of the complete DNA polymerase gene or directly from the cloned inteins genes. Both recombinant inteins exhibit endonuclease activity, with an optimal temperature of 70°C. The Tfu pol-1 intein, which belongs to the Psp KOD pol-1 allelic family, recognizes and cleaves a minimal sequence of 16 base pairs (bp) on supercoiled DNA with either Mn 2؉ or Mg 2؉ as cofactor. It cleaves linear DNA only with Mn 2؉ and requires a 19-bp minimal recognition sequence. The Tfu pol-2 intein, which belongs to the Tli pol-2 allelic family, is a highly active homing endonuclease using Mg 2؉ as cofactor. Its minimal recognition and cleavage site is 21 bp long either on linear or circular DNA substrates. Its endonuclease activity is strongly inhibited by the 3 digestion product, which remains bound to the enzyme after the cleavage reaction. According to current nomenclature, these endonucleases were named PI-TfuI and PI-TfuII. These two inteins thus exhibit different requirements for metal cofactor and substrate topology as well as different mechanism of action.Since the first report of a protein splicing element as an in-frame insertion in the VMA gene of Saccharomyces cerevisiae (1, 2), over 80 putative inteins have been identified.

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“…Possible reasons for this might be that the distance and/or angle between the cofactor binding amino acid residues in the modified enzyme are not optimal. These factors have been shown to play a role in the effects of metal cofactors on the catalysis of other enzymes (e.g., EcoRV restriction endonuclease) (Vipond et al, 1996;Bock et al, 1999). In addition to Mg 2+ , Mn 2+ , and Zn 2+ , other divalent cations support catalysis to some extent, including Ca 2+ and Co 2+ .…”

Section: Alterations In the Hxh[dn]d Motif Shift Preference For The Dmentioning

confidence: 99%

From Amino Acid to Glucosinolate Biosynthesis: Protein Sequence Changes in the Evolution of Methylthioalkylmalate Synthase in Arabidopsis

2011

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Methylthioalkylmalate synthase (MAM) catalyzes the committed step in the side chain elongation of Met, yielding important precursors for glucosinolate biosynthesis in Arabidopsis thaliana and other Brassicaceae species. MAM is believed to have evolved from isopropylmalate synthase (IPMS), an enzyme involved in Leu biosynthesis, based on phylogenetic analyses and an overlap of catalytic abilities. Here, we investigated the changes in protein structure that have occurred during the recruitment of IPMS from amino acid to glucosinolate metabolism. The major sequence difference between IPMS and MAM is the absence of 120 amino acids at the C-terminal end of MAM that constitute a regulatory domain for Leu-mediated feedback inhibition. Truncation of this domain in Arabidopsis IPMS2 results in loss of Leu feedback inhibition and quaternary structure, two features common to MAM enzymes, plus an 8.4-fold increase in the k cat /K m for a MAM substrate. Additional exchange of two amino acids in the active site resulted in a MAM-like enzyme that had little residual IPMS activity. Hence, combination of the loss of the regulatory domain and a few additional amino acid exchanges can explain the evolution of MAM from IPMS during its recruitment from primary to secondary metabolism.

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An Isoleucine to Leucine Mutation that Switches the Cofactor Requirement of the EcoRV Restriction Endonuclease from Magnesium to Manganese

Cited by 47 publications

References 32 publications

Amino Acid Residues in Both the Protein Splicing and Endonuclease Domains of the PI-SceI Intein Mediate DNA Binding

Amino Acid Residues in Both the Protein Splicing and Endonuclease Domains of the PI-SceI Intein Mediate DNA Binding

Inteins of Thermococcus fumicolans DNA Polymerase Are Endonucleases with Distinct Enzymatic Behaviors

From Amino Acid to Glucosinolate Biosynthesis: Protein Sequence Changes in the Evolution of Methylthioalkylmalate Synthase in Arabidopsis

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