4AHQ: Crystal Structure Of N-acetylneuraminic Acid Lyase Mutant K165c From Staphylococcus Aureus

Chemical modification has been used to introduce the unnatural amino acid gamma-thialysine in place of the catalytically important Lys165 in the enzyme N-acetylneuraminic acid lyase (NAL). The Staphylococcus aureus nanA gene, encoding NAL, was cloned and expressed in E. coli. The protein, purified in high yield, has all the properties expected of a class I NAL. The S. aureus NAL which contains no natural cysteine residues was subjected to site-directed mutagenesis to introduce a cysteine in place of Lys165 in the enzyme active site. Subsequently chemical mutagenesis completely converted the cysteine into gamma-thialysine through dehydroalanine (Dha) as demonstrated by ESI-MS. Initial kinetic characterisation showed that the protein containing gamma-thialysine regained 17 % of the wild-type activity. To understand the reason for this lower activity, we solved X-ray crystal structures of the wild-type S. aureus NAL, both in the absence of, and in complex with, pyruvate. We also report the structures of the K165C variant, and the K165-gamma-thialysine enzyme in the presence, or absence, of pyruvate. These structures reveal that gamma-thialysine in NAL is an excellent structural mimic of lysine. Measurement of the pH-activity profile of the thialysine modified enzyme revealed that its pH optimum is shifted from 7.4 to 6.8. At its optimum pH, the thialysine-containing enzyme showed almost 30 % of the activity of the wild-type enzyme at its pH optimum. The lowered activity and altered pH profile of the unnatural amino acid-containing enzyme can be rationalised by imbalances of the ionisation states of residues within the active site when the pK(a) of the residue at position 165 is perturbed by replacement with gamma-thialysine. The results reveal the utility of chemical mutagenesis for the modification of enzyme active sites and the exquisite sensitivity of catalysis to the local structural and electrostatic environment in NAL.
PDB ID: 4AHQDownload
MMDB ID: 106865
PDB Deposition Date: 2012/2/6
Updated in MMDB: 2013/03
Experimental Method:
x-ray diffraction
Resolution: 1.95  Å
Source Organism:
Similar Structures:
Biological Unit for 4AHQ: tetrameric; determined by author and by software (PISA)
Molecular Components in 4AHQ
Label Count Molecule
Proteins (4 molecules)
N-acetylneuraminate Lyase(Gene symbol: SAOUHSC_00295)
Molecule annotation
* Click molecule labels to explore molecular sequence information.

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