Structural Insights into the Recovery of Aldolase Activity in N‐Acetylneuraminic Acid Lyase by Replacement of the Catalytically Active Lysine with γ‐Thialysine by Using a Chemical Mutagenesis Strategy

Abstract

Chemical modification has been used to introduce the unnatural amino acid γ-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 γ-thialysine through dehydroalanine (Dha) as demonstrated by ESI-MS. Initial kinetic characterisation showed that the protein containing γ-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-γ-thialysine enzyme in the presence, or absence, of pyruvate. These structures reveal that γ-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 pKa of the residue at position 165 is perturbed by replacement with γ-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.

Extending enzyme molecular recognition with an expanded amino acid alphabet

“…1A). The substrate specificity and stereochemistry of this enzyme is highly malleable through protein engineering and directed evolution (34,35), and we have shown that an enzyme bearing the Nca γ-thialysine (2-aminoethyl cysteine) in place of the catalytic Lys-165 retains activity (36). Here we have explored the effect of introducing Ncas throughout the active site on the reaction catalyzed.…”

“…Incorporation was carried out on a small (2.5 mg) scale under denaturing conditions to ensure the cysteine residue was accessible for modification before the protein was refolded. This procedure was previously shown to generate active, modified, refolded enzyme (36). The conversions of Cys→Dha and Dha→Nca were both monitored by ESI mass spectrometry (Figs.…”

“…3). The equivalent positions in the S. aureus NAL based on structural alignment were then mutated into cysteine residues by site-directed mutagenesis, and expressed and purified as previously described (36). The Ncas were then incorporated by first converting the cysteine to dehydroalanine (Dha) using 2,5-dibromohexan-1,6-diamide and then Michael addition with 13 different thiols resulting in installation of 13 different Ncas at each of the 12 positions (27) (Fig.…”

“…2). The chemical modification procedure to introduce the Nca relies on the conversion of a cysteine into dehydroalanine by a bis-alkylation/ elimination sequence (27,36), followed by Michael addition of a thiol to introduce the new Nca side chain. The S. aureus NAL naturally contains no cysteines, making it an ideal target protein for chemical introduction of Ncas (36).…”

“…The chemical modification procedure to introduce the Nca relies on the conversion of a cysteine into dehydroalanine by a bis-alkylation/ elimination sequence (27,36), followed by Michael addition of a thiol to introduce the new Nca side chain. The S. aureus NAL naturally contains no cysteines, making it an ideal target protein for chemical introduction of Ncas (36). The residues chosen for modification were picked based on their proximity to the aldehyde substrate binding pocket in the previously solved structure of an Escherichia coli NAL variant in complex with 4-epi N-acetylneuraminic acid (PDB ID code 4BWL) (37) (Fig.…”