The creation of artificial enzymes to catalyze desired reactions is a major goal of computational protein design. However, de novo enzymes display low catalytic efficiencies, requiring the introduction of activity-enhancing active site and distal mutations through directed evolution. A better understanding of how mutations introduced by directed evolution contribute to increased enzymatic activity will guide the development of design methods such that efficient enzymes can be designed de novo. Here, we evaluate the structural, functional, and dynamical impacts of active site and distal mutations introduced by directed evolution of the de novo retro-aldolase RA95, an enzyme that presents an important case study in enzyme design due to the significant structural remodelling that was observed during evolution. We observe that the variant RA95-Core, containing only active site mutations introduced by directed evolution, displays activity within one order of magnitude of the fully evolved variant. This suggests that computational enzyme design methods can be improved to create much more efficient enzymes than what was previously achieved in RA95. However, structural changes induced by distal mutations prevent computational recapitulation of the evolved active site on the original design template, indicating that the optimized active site identified through directed evolution could not have been designed de novo using current design methodologies. We suggest strategies for the incorporation of backbone remodelling into design procedures that would allow recapitulation of the evolved retro-aldolase active site, as well as the de novo design of highly efficient enzymes without the need for optimization by directed evolution.

Call number: LD2668 .T4 1978 C51 / Master of Science

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The structure of the dihydrolipoamide acetyltransferase (E2p) component of the pyruvate dehydrogenase complex from <i>Escherichia coli</i> and its role in catalysis were studied by the combined approaches of protein engineering, limited proteolysis and <SUP>1</SUP>H-n.m.r. spectroscopy. Genetic reconstruction of the E2p component (performed elsewhere) produced a series of mutant complexes assembled around E2p chains which contain only a single lipoyl domain and an associated (alanine+proline)-rich linker region of gradually diminishing lengths (32, 20, 13, 7 and 1 residue(s), respectively, in the pGS110-,pGS156-,pGS186 ,pGS187- and pGS188-encoded complexes). When this region was shortened to 13 residues or less, the system of active-site coupling in the enzyme complex was dramatically impaired, although the individual enzyme activities were unaffected. The role of the (alanine+proline)-rich region in facilitating moment of the lipoyl domains in catalysis was thus established. The (alanine+proline)-rich regions of the wild-type E2p chains had previously been conjectured to be the source of the unexpectedly sharp resonances in the <SUP>1</SUP>H-n.m.r. spectrum of the enzyme complex, and hence to be conformationally mobile. Examination of the genetically restructured complexes by <SUP>1</SUP>H-n.m.r. spectroscopy revealed that the intensity of the sharp peaks in the spectra correlated well with the length of the (alanine+proline)-rich region in each complex. Furthermore, resonances from a single histidine residue engineered into the (alanine+proline)-rich region of a pGS110-encoded E2p chain was clearly visible in the <SUP>1</SUP>H-n.m.r. spectrum of the resulting enzyme complex. These experiments proved unequivocally that the (alanine+proline)-rich regions are conformationally mobile. The <SUP>1</SUP>H-n.m.r. spectra of the mutant complexes with the most severe deletions in the E2p chains differed from those of the wild-type and pGS110-encoded complexes in that they displayed a novel sharp peak which was not initially apparent in the spectra of the parent assemblies. This resonance was tentatively assigned to another, shorter (alanine+proline)-rich sequence in the E2p chain, which separates the dihydrolipoamide dehydrogenase (E3)-binding and inner-core domains in the <i>C</i>-terminal half of the molecule. It is likely therefore that this sequence is also conformationally flexible. Antibodies against a synthetic peptide with the sequence of the long (alanine+proline)-rich region of the pGS110-encoded E2p chain were raised elsewhere. Binding of the Fab fragments of these antibodies to the pGS110-encoded complex was found to inhibit the overall complex activity even though the activities of the three component enzymes were not affected. Antibody binding was shown to prevent both the reductive acetylation of the lipoyl domains at the pyruvate decarboxylase (E1p) active site and the transfer of acetyl groups between adjacent lipoyl domains, demonstrating the role of the (alanine+proline)-rich sequence in the mechanism of substrate transfer between active sites. A detailed study of the conformation of the (alanine+proline)-rich regions was also undertaken. Synthetic peptides were obtained with sequences identical to the central and innermost such regions of the wild-type E2p chain. The conformation of these peptides in aqueous solution was studied by circular dichroism, <SUP>1</SUP>H-n.m.r. and <SUP>13</SUP>C-n.m.r. spectroscopy. Relaxation time and nOe data pointed to an extended conformation for the peptides, a structure enforced by the predominantly <i>trans</i> Ala-Pro peptide bond. The functional consequences of this conformation and the role of these sequences in the structure and the function of the enzyme complex are discussed.

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