Directed evolution was used to change the substrate specificity of aspartate aminotransferase. A mutant enzyme with 17 amino acid substitutions was generated that shows a 2.1 ؋ 10 6 -fold increase in the catalytic efficiency (k cat /K m ) for a non-native substrate, valine. The absorption spectrum of the bound coenzyme, pyridoxal 5-phosphate, is also changed significantly by the mutations. Interestingly, only one of the 17 residues appears to be able to contact the substrate, and none of them interact with the coenzyme. The three-dimensional structure of the mutant enzyme complexed with a valine analog, isovalerate (determined to 2.4-Å resolution by x-ray crystallography), provides insights into how the mutations affect substrate binding. The active site is remodeled; the subunit interface is altered, and the enzyme domain that encloses the substrate is shifted by the mutations. The present results demonstrate clearly the importance of the cumulative effects of residues remote from the active site and represent a new line of approach to the redesign of enzyme activity.

The substrate specificity of aspartate aminotransferase was successfully modified by directed molecular evolution using a combination of DNA shuff ling and selection in an auxotrophic Escherichia coli strain. After five rounds of selection, one of the evolved mutants showed a 10 5 -fold increase in the catalytic efficiency (k cat ͞K m ) for ␤-branched amino and 2-oxo acids and a 30-fold decrease in that for the native substrates compared with the wild-type enzyme. The mutant had 13 amino acid substitutions, 6 of which contributed 80-90% to the total effect. Five of these six substitutions were conserved among the five mutants that showed the highest activity for ␤-branched substrates. Interestingly, only one of the six functionally important residues is located within a distance of direct interaction with the substrate, supporting the idea that rational design of the substrate specificity of an enzyme is very difficult. The present results show that directed molecular evolution is a powerful technique for enzyme redesign if an adequate selection system is applied.To alter the substrate specificity of an existing enzyme is worth trying because it provides not only efficient catalysts with designed substrate specificity but also valuable information on the mechanism of substrate recognition. Despite numerous studies to engineer enzymes over the past decade, only a few of them have reported ϳ10 3 -fold enhancements in the catalytic efficiency for the nonnative substrates (1-3). Recently, strategies based on directed molecular evolution have created macromolecules with novel properties (4-10). A method called DNA shuffling has proved promising as a technique to introduce random mutations and recombinations among a population of mutant genes (11-14). A major obstacle that restricts the utility of these methods in redesigning enzymes is the technical difficulty of establishing a rapid and sensitive system to screen Ͼ10 4 mutant enzymes for the desired activity. Chromogenic substrates were successfully used for a screening system utilizing 96-well plates and a plate reader (6) or a visual screening of the colored colonies (14). These substrates are, however, available only for a limited range of reactions. An ideal screening system should be a convenient and reliable one, of which the sensitivity can be adjusted depending on the progress of the selection steps. One possible system is to select mutant enzymes that can reverse the phenotype of a bacterial strain that is deficient in an enzyme with the desired activity. The sensitivity or stringency of the selection can be adjusted by supplementing the medium with an adequate amount of the substrate or product.Aminotransferases catalyze amino group transfer between amino acids and 2-oxo acids and play central roles in amino acid metabolism in organisms ranging from bacteria to mammals. The structure (15, 16) and reaction mechanism (17) of aspartate 2-oxoglutarate aminotransferase (AspAT) have been studied extensively. Among the natural amino acids, AspAT from Esch...

The whole-genome sequencing of an extreme thermophile, Thermus thermophilus, is now in progress. Like other genome projects, major concern is shifting from the sequence itself to post-sequencing research such as functional or structural genomics. Under such circumstances, the demand for convenient genetic-engineering tools is increasing. In this study we have increased the thermostability of a kanamycin-resistance gene product using strategies based on directed evolution in T. thermophilus to the upper limit of its growth temperature. The most thermostable mutant has 19 amino-acid substitutions, whereby the thermostability is increased by 20 degrees C, but the enzymatic activity is not significantly changed. Most of the mutated residues are located on the surface of the protein molecule, and, interestingly, five of the 19 substitutions are those to proline residues. The evolved kanamycin-resistance gene products could be used as selection markers at the optimum growth temperature of T. thermophilus. The development of such a convenient genetic-engineering tool would facilitate post-sequencing research on T. thermophilus.

Genes of an extremely thermophilic bacterium, Thermus thermophilus, were disrupted by homologous recombination using a recently developed, thermostable kanamycinresistant marker. First, the trpE gene was disrupted with various constructions of DNA. The transformation efficiency was exponentially increased as the length of the homologous regions flanking the marker gene increased above the minimum length (200^300 bp). We then disrupted five genes of the nucleotide excision repair system and examined their phenotypes. The convenience and high reliability of this method should prompt its application to the high-throughput systematic disruption of the genes of this thermophilic bacterium. ß

We recently identified a previously unidentified sphingosine-1-phosphate (S1P) signaling mechanism that stimulates production of a key innate immune element, cathelicidin antimicrobial peptide (CAMP), in mammalian cells exposed to external perturbations, such as UVB irradiation and other oxidative stressors that provoke subapoptotic levels of endoplasmic reticulum (ER) stress, independent of the well-known vitamin D receptor-dependent mechanism. ER stress increases cellular ceramide and one of its distal metabolites, S1P, which activates NF-κB followed by C/EBPα activation, leading to CAMP production, but in a S1P receptor-independent fashion. We now show that S1P activates NF-κB through formation of a previously unidentified signaling complex, consisting of S1P, TRAF2, and RIP1 that further associates with three stress-responsive proteins; i.e., heat shock proteins (GRP94 and HSP90α) and IRE1α. S1P specifically interacts with the N-terminal domain of heat shock proteins. Because this ER stress-initiated mechanism is operative in both epithelial cells and macrophages, it appears to be a universal, highly conserved response, broadly protective against diverse external perturbations that lead to increased ER stress. Finally, these studies further illuminate how ER stress and S1P orchestrate critical stress-specific signals that regulate production of one protective response by stimulating production of the key innate immune element, CAMP.ER stress | sphingosine-1-phosphate | heat shock protein 90 | cathelicidin | innate immunity