The high-resolution X-ray structure of wild-type staphylococcal nuclease (E43 SNase) suggests that Glu 43 acts a general basic catalyst to assist the attack of water on a phosphodiester substrate [Loll, P., & Lattman, E. E. (1989) Proteins: Struct., Funct., Genet. 5, 183]. Glu 43 is located at the base of the solvent-exposed and conformationally mobile omega-loop in the active site of E43 SNase having the sequence Glu43-Thr44-Lys45-His46-Pro47-Lys48- Lys49-Gly50-Val51-Glu52, where the gamma-carboxylate of Glu 52 is hydrogen bonded to the amide hydrogen of Glu 43. With a metabolic selection for SNase activity produced in an Escherichia coli host, we detected an unexpected deletion of residues 44-49 of the omega-loop of E43 SNase in cassette mutagenesis experiments designed to randomize codons 44 and 45 in the omega-loop and increase the activity of the previously described E43D mutation (D43 SNase). A high-resolution X-ray structure of D43 SNase has revealed that the E43D substitution significantly changes the structure of the omega-loop, reduces the interaction of the essential Ca2+ ion with its active-site ligands, and diminishes the network of hydrogen-bonded water molecules in the active site [Loll, P., & Lattman, E. E. (1990) Biochemistry 29, 6866]. This deletion of six amino acids from the omega-loop generates a protein (E43 delta SNase) having a partially solvent-exposed, surface beta-turn with the sequence Glu43-Gly50-Val51-Glu52; the structure of this beta-turn is addressed in the following article [Baldisseri et al. (1991) Biochemistry (following paper in this issue)].(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 4243, 4244 Extra Details:
ID: c6jivvr74
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:25 p.m.
Version: 1
Number of data points | 7 |
Proteins | Thermonuclease ; Thermonuclease |
Unique complexes | 2 |
Assays/Quantities/Protocols | Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O ; Derived Quantity: ddG_H2O |
Libraries | Mutations for sequence ATSTKKLHKEPATLIKAIDGDTVKLMYKGQPMTFRLLLVDTPETKHPKKGVEKYGPEASAFTKKMVENAKKIEVEFDKGQRTDKYGRGLAYIYADGKMVNEALVRQGLAKVAYVYKPNNTHEQHLRKSEAQAKKEKLNIWSEDNADSGQ |
Colors: | D | E | R | H | K | S | T | N | Q | A | V | I | L | M | F | Y | W | C | G | P |
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Percent Identity | Matching Chains | Protein | Accession | Entry Name |
---|---|---|---|---|
100.0 | Thermonuclease | P00644 | NUC_STAAU | |
99.3 | Thermonuclease | Q5HHM4 | NUC_STAAC | |
99.1 | Thermonuclease | Q99VJ0 | NUC_STAAM | |
99.1 | Thermonuclease | Q7A6P2 | NUC_STAAN | |
99.3 | Thermonuclease | Q6GB41 | NUC_STAAS | |
99.3 | Thermonuclease | Q8NXI6 | NUC_STAAW | |
99.3 | Thermonuclease | Q6GIK1 | NUC_STAAR |