Deletion of the omega-loop in the active site of staphylococcal nuclease. 1. Effect on catalysis and stability.
Abstract
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:
Submission Details
ID: c6jivvr74
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:25 p.m.
Version: 1
Publication Details
Poole LB;Loveys DA;Hale SP;Gerlt JA;Stanczyk SM;Bolton PH,Biochemistry (1991) Deletion of the omega-loop in the active site of staphylococcal nuclease. 1. Effect on catalysis and stability. PMID:2015219Additional Information
Study Summary
| 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 |
Structure view and single mutant data analysis
Study data
| Colors: | D | E | R | H | K | S | T | N | Q | A | V | I | L | M | F | Y | W | C | G | P |
|---|
Data Distribution
Studies with similar sequences (approximate matches)
Correlation with other assays (exact sequence matches)
Relevant PDB Entries
| Structure ID | Release Date | Resolution | Structure Title |
|---|---|---|---|
| 4WRD | 2014-10-23T00:00:00+0000 | 1.65 | Crystal structure of Staphylcoccal nulease variant Delta+PHS V66E L125E at cryogenic temperature |
| 2LKV | 2011-10-21T00:00:00+0000 | 0 | Staphylococcal Nuclease PHS variant |
| 2M00 | 2012-10-14T00:00:00+0000 | 0 | Solution structure of staphylococcal nuclease E43S mutant in the presence of ssDNA and Cd2+ |
| 2OXP | 2007-02-20T00:00:00+0000 | 2.0 | Crystal Structure of Staphylococcal Nuclease mutant V66D/P117G/H124L/S128A |
| 3D4W | 2008-05-15T00:00:00+0000 | 1.9 | Crystal structure of Staphylococcal nuclease variant Delta+PHS A109R at cryogenic temperature |
| 3D8G | 2008-05-23T00:00:00+0000 | 1.99 | Crystal structure of Staphylococcal nuclease variant Delta+PHS I72R at cryogenic temperature |
| 3MVV | 2010-05-04T00:00:00+0000 | 1.72 | Crystal structure of Staphylococcal nuclease variant Delta+PHS F34A at cryogenic temperature |
| 3QOJ | 2011-02-10T00:00:00+0000 | 1.6 | Cryogenic structure of Staphylococcal nuclease variant D+PHS/V23K |
| 3QOL | 2011-02-10T00:00:00+0000 | 1.9 | Crystal structure of Staphylococcal nuclease variant D+PHS/V23E at pH 6 determined at 100 K |
| 3R3O | 2011-03-16T00:00:00+0000 | 1.9 | Crystal structure of Staphylococcal nuclease variant Delta+PHS T62A at cryogenic temperature and with high redundancy |
Relevant UniProtKB Entries
| Percent Identity | Matching Chains | Protein | Accession | Entry Name |
|---|---|---|---|---|
| 99.3 | Thermonuclease | Q6GIK1 | NUC_STAAR | |
| 99.3 | Thermonuclease | Q8NXI6 | NUC_STAAW | |
| 99.3 | Thermonuclease | Q6GB41 | NUC_STAAS | |
| 99.1 | Thermonuclease | Q7A6P2 | NUC_STAAN | |
| 99.1 | Thermonuclease | Q99VJ0 | NUC_STAAM | |
| 99.3 | Thermonuclease | Q5HHM4 | NUC_STAAC | |
| 100.0 | Thermonuclease | P00644 | NUC_STAAU |