Ancestral sequence reconstruction produces thermally stable enzymes with mesophilic enzyme-like catalytic properties

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.

Evolution of Protein Structure and Stability in Global Warming

“…Thus, use of ASR pointed to progenotes that were even more thermophilic than the Archaea , consistent with the high heat of the primordial environment. Similar studies with many other enzymes have further confirmed the conclusion that early organisms were indeed endowed with superior thermoresistance, adding confidence to ASR [ 50 , 59 , 60 , 61 , 62 , 63 ]. To the molecular biologist, the ASR studies provide an opportunity to understand the roles of specific amino acids in thermophilia; however, this requires analyses of additional proteins and determination of their structures.…”

State-of-the-Art Biocatalysis

“…To this effect, Furukawa et al have identified an ancestor of 3-isopropylmalate dehydrogenase (IPMDH), a key enzyme in the biosynthesis of leucine, which offers improvements in its stability and activity over extant IPMDH enzymes from present-day organisms through construction and analysis of a phylogenetic tree. 72 Following inference and identification of two ancestral protein sequences, dubbed ancIPMDH-IQ and ancIPMDH-ML, the group successfully expressed each protein in E. coli and, after isolating the enzymes for further investigation, discovered they provided increased thermal stability and improved catalytic activity at low temperatures compared to their modern homologues. 72 This work demonstrates just one of many potential uses for ancestral protein reconstruction, as other reports describe how ancestral proteins might possess higher degrees of substrate promiscuity compared to their modern offspring, thus offering potentially valuable characteristics to organic chemists seeking diverse and novel bond-forming activity.…”

Catalytic mechanism of ancestral L-lysine oxidase assigned by sequence data mining

“…Here, ASR is a sequence-based protein redesign method which can generate ancestral proteins located at each node of the phylogenetic tree (20,21). Ancestral proteins often have desirable properties for use in practical applications, such as high thermostability (22,23) and broad substrate selectivity (24). Therefore, ASR is currently being adopted as a tool for protein engineering (25).…”