Abstract
Enzymes have high catalytic efficiency and low environmental impact, and are therefore potentially useful tools for various industrial processes. Crucially, however, natural enzymes do not always have the properties required for specific processes. It may be necessary, therefore, to design, engineer, and evolve enzymes with properties that are not found in natural enzymes. In particular, the creation of enzymes that are thermally stable and catalytically active at low temperature is desirable for processes involving both high and low temperatures. In the current study, we designed two ancestral sequences of 3-isopropylmalate dehydrogenase by an ancestral sequence reconstruction technique based on a phylogenetic analysis of extant homologous amino acid sequences. Genes encoding the designed sequences were artificially synthesized and expressed in Escherichia coli. The reconstructed enzymes were found to be slightly more thermally stable than the extant thermophilic homologue from Thermus thermophilus. Moreover, they had considerably higher low-temperature catalytic activity as compared with the T. thermophilus enzyme. Detailed analyses of their temperature-dependent specific activities and kinetic properties showed that the reconstructed enzymes have catalytic properties similar to those of mesophilic homologues. Collectively, our study demonstrates that ancestral sequence reconstruction can produce a thermally stable enzyme with catalytic properties adapted to low-temperature reactions.
Highlights
Enzymes have high catalytic efficiency and low environmental impact, and are potentially useful tools for various industrial processes
Cold-active enzymes isolated from psychrophilic organisms are often less stable but catalytically more active at low temperatures compared with their mesophilic and thermophilic counterparts[6,7,8,9,10,11], certain cold-active enzymes in nature are very s table[12,13]
The design method consists of three steps: (1) inference of an ancestral sequence based on a comparison of homologous amino acid sequences; (2) artificial synthesis of a gene encoding the inferred amino acid sequence; and (3) expressing the gene in a host organism such as Escherichia coli
Summary
Enzymes have high catalytic efficiency and low environmental impact, and are potentially useful tools for various industrial processes. The reconstructed enzymes were found to be slightly more thermally stable than the extant thermophilic homologue from Thermus thermophilus They had considerably higher low-temperature catalytic activity as compared with the T. thermophilus enzyme. ASR can be a very powerful tool for accessing amino acid substitutions remote from the active site, which influence activity and thermostability[27,28] This method does not require knowledge about the three-dimensional structure or design principles of the protein. The design method consists of three steps: (1) inference of an ancestral sequence based on a comparison of homologous amino acid sequences; (2) artificial synthesis of a gene encoding the inferred amino acid sequence; and (3) expressing the gene in a host organism such as Escherichia coli Proteins reconstructed in this way are often highly thermostable[29,30,31,32]. By substituting putative ancestral amino acids into natural enzymes, the thermostability of a natural protein is highly likely to be increased without compromising the catalytic a ctivity[33,34,35,36,37]
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