2WPB

Crystal structure of the E192N mutant of E. Coli N-acetylneuraminic acid lyase in complex with pyruvate and the inhibitor (2R,3R)-2,3,4- trihydroxy-N,N-dipropylbutanamide in space group P21 crystal form I

Summary for 2WPB

• Entry DOI: 10.2210/pdb2wpb/pdb
• Related: 1FDY 1FDZ 1HL2 1NAL 2WKJ 2WNN 2WNQ 2WNZ 2WO5 2WSG
• Descriptor: N-ACETYLNEURAMINATE LYASE, (2R,3R)-2,3,4-TRIHYDROXY-N,N-DIPROPYLBUTANAMIDE (3 entities in total)
• Functional Keywords: substrate specificity, carbohydrate metabolism, protein engineering, lyase, aldolase, schiff base
• Biological source: ESCHERICHIA COLI
• Cellular location: Cytoplasm: P0A6L4
• Total number of polymer chains: 4
• Total formula weight: 135430.69

Authors

Campeotto, I.,Bolt, A.H.,Harman, T.A.,Trinh, C.H.,Dennis, C.A.,Phillips, S.E.V.,Pearson, A.R.,Nelson, A.,Berry, A. (deposition date: 2009-08-03, release date: 2010-08-25, Last modification date: 2023-12-20)

Primary citation

Campeotto, I.,Bolt, A.H.,Harman, T.A.,Dennis, C.A.,Trinh, C.H.,Phillips, S.E.V.,Nelson, A.,Pearson, A.R.,Berry, A.
Structural Insights Into Substrate Specificity in Variants of N-Acetylneuraminic Acid Lyase Produced by Directed Evolution.
J.Mol.Biol., 404:56-, 2010

PubMed 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 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.

PubMed: 20826162
DOI: 10.1016/J.JMB.2010.08.008

Experimental method

X-RAY DIFFRACTION (2.05 Å)