Structural Insights into Substrate Specificity in Variants of N-Acetylneuraminic Acid Lyase Produced by Directed Evolution

Abstract

The substrate specificity of Escherichia coli N-acetylneuraminic acid lyase was previously switched from the natural condensation of pyruvate with N-acetylmannosamine, yielding N-acetylneuraminic acid, to the aldol condensation generating N-alkylcarboxamide analogues of N-acetylneuraminic acid. This was achieved by a single mutation of Glu192 to Asn. In order to analyze the structural changes involved and to more fully understand the basis of this switch in specificity, we have isolated all 20 variants of the enzyme at position 192 and determined the activities with a range of substrates. We have also determined five high-resolution crystal structures: the structures of wild-type E. coli N-acetylneuraminic acid lyase in the presence and in the absence of pyruvate, the structures of the E192N variant in the presence and in the absence of pyruvate, and the structure of the E192N variant in the presence of pyruvate and a competitive inhibitor (2R,3R)-2,3,4-trihydroxy-N,N-dipropylbutanamide. All structures were solved in space group P21 at resolutions ranging from 1.65 Å to 2.2 Å. A comparison of these structures, in combination with the specificity profiles of the variants, reveals subtle differences that explain the details of the specificity changes. This work demonstrates the subtleties of enzyme–substrate interactions and the importance of determining the structures of enzymes produced by directed evolution, where the specificity determinants may change from one substrate to another.

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

“…The crystal structure of the S. aureus NAL shows that the enzyme adopts a TIM-(β/α) 8 -barrel tertiary structure identical to that previously observed for the E. coli (PDB ID: 2WO5)[29] and Haemophilus influenzae (PDB ID: 1F5Z)[30] NALs, with RMSD values of 1.36 and 0.84 Å for alignments of the subunit alpha-carbons of the S. aureus structure with the E. coli and H. influenzae enzymes, respectively. Comparison of the E. coli (PDB ID: 2WNN)[29] and S. aureus (4AH7) enzymes in complex with pyruvate showed that the pyruvate is covalently linked to the enzyme as a Schiff base and makes identical interactions with the backbone amides of Ser48 and Ser49 and also with the side-chain hydroxyl group of Ser49 (equivalent to Ser47 and Thr48 in the E. coli enzyme; Figure 3 A and B).…”

Development of an organo- and enzyme-catalysed one-pot, sequential three-component reaction

“…The structure of complexes between the E192N NAL variant, pyruvate and a substrate analogue suggested that a hydroxyl group  to the aldehyde may not be essential for binding. 8 Accordingly, it was hoped that the aldol products 6 would be viable substrates for the E192N variant, enabling the synthesis of the final products 7 by enzymic addition of pyruvate. The condensation of the aldehyde 4, acetaldehyde and pyruvate (5), to give 7, would constitute the first example of a three-component reaction in which two carboncarbon bond-forming steps were catalysed using the specific combination of an organocatalyst and an enzyme.…”

Extending enzyme molecular recognition with an expanded amino acid alphabet

“…Crystallization conditions were as previously described (36,53). Diffraction data for the Phe190Dpc structure were collected from a single crystal at the Diamond Light Source macromolecular crystallography beam line I04-1 flash-cooled to 100 K. Data processing and refinement were carried out as previously described (36).…”