N-Acetylneuraminic acid lyase (NAL) is a Class I aldolase
that catalyzes the reversible condensation of pyruvate with N-acetyl-d-mannosamine (ManNAc) to yield the sialic
acid N-acetylneuraminic acid (Neu5Ac). Aldolases
are finding increasing use as biocatalysts for the stereospecific
synthesis of complex molecules. Incomplete understanding of the mechanism
of catalysis in aldolases, however, can hamper development of new
enzyme activities and specificities, including control over newly
generated stereocenters. In the case of NAL, it is clear that the
enzyme catalyzes a Bi-Uni ordered condensation reaction in which pyruvate
binds first to the enzyme to form a catalytically important Schiff
base. The identity of the residues required for catalysis of the condensation
step and the nature of the transition state for this reaction, however,
have been a matter of conjecture. In order to address, this we crystallized
a Y137A variant of the E. coli NAL in the presence
of Neu5Ac. The three-dimensional structure shows a full length sialic
acid bound in the active site of subunits A, B, and D, while in subunit
C, discontinuous electron density reveals the positions of enzyme-bound
pyruvate and ManNAc. These ‘snapshot’ structures, representative
of intermediates in the enzyme catalytic cycle, provided an ideal
starting point for QM/MM modeling of the enzymic reaction of carbon–carbon
bond formation. This revealed that Tyr137 acts as the proton donor
to the aldehyde oxygen of ManNAc during the reaction, the activation
barrier is dominated by carbon–carbon bond formation, and proton
transfer from Tyr137 is required to obtain a stable Neu5Ac-Lys165
Schiff base complex. The results also suggested that a triad of residues,
Tyr137, Ser47, and Tyr110 from a neighboring subunit, are required
to correctly position Tyr137 for its function, and this was confirmed
by site-directed mutagenesis. This understanding of the mechanism
and geometry of the transition states along the C–C bond-forming
pathway will allow further development of these enzymes for stereospecific
synthesis of new enzyme products.
This review focuses on the directed evolution of aldolases with synthetically useful properties. Directed evolution has been used to address a number of limitations associated with the use of wild-type aldolases as catalysts in synthetic organic chemistry. The generation of aldolase enzymes with a modified or expanded substrate repertoire is described. Particular emphasis is placed on the directed evolution of aldolases with modified stereochemical properties: such enzymes can be useful catalysts in the stereoselective synthesis of biologically active small molecules. The review also describes some of the fundamental insights into mechanistic enzymology that directed evolution can provide.
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.
The bisindolylmaleimides are selective protein kinase inhibitors that can adopt two limiting diastereomeric (syn and anti) conformations. The configurational stability of a range of substituted and macrocyclic bisindolylmaleimides was investigated by using appropriate techniques. With unconstrained bisindolylmaleimides, the size of the 2-indolyl substituents was found to affect configurational stability, though not sufficiently to allow atropisomeric bisindolylmaleimides to be obtained. However, with a tether between the two indole nitrogen atoms in place, the steric effect of 2-indolyl substituents was greatly exaggerated, leading to large differences in configurational stability. The rate of interconversion of the syn and anti conformers varied by over twenty orders of magnitude through substitution of a bisindolylmaleimide ring system, which was constrained within a macrocyclic ring. Indeed, the first examples of configurationally stable atropisomeric bisindolylmaleimides are reported; the half-life for epimerisation of these compounds at room temperature was estimated to be >10(7) years.
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