Structural insights into substrate specificity in variants of N-acetylneuraminic Acid lyase produced by directed evolution.
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 P2(1) 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.
Campeotto
Ivan
I
Astbury Center for Structural Molecular Biology, Garstang Building, University of Leeds, Leeds LS2 9JT, UK.
Bolt
Amanda H
AH
Harman
Thomas A
TA
Dennis
Caitriona
C
Trinh
Chi H
CH
Phillips
Simon E V
SE
Nelson
Adam
A
Pearson
Arwen R
AR
Berry
Alan
A
D019943:Amino Acid Substitution; D018360:Crystallography, X-Ray; D019020:Directed Molecular Evolution; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D007652:Oxo-Acid-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D050505:Mutant Proteins; D007652:Oxo-Acid-Lyases; C018728:N-acetylneuraminate lyase
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