Thrice upon a time: The repeated emergence of a novel enzymatic function from an evolvable protein scaffold

Conference Dates

September 15-19, 2019


Understanding the emergence of new protein functions from their ancestors is a long-standing challenge in biology and biotechnology; many questions remain unanswered. How can one protein scaffold support multiple distinct functions? How are diverse functions of a superfamily connected? How are major functional switches achieved? Large-scale experimental approaches that systematically determine the activity profiles across enzyme superfamilies have now begun to provide comprehensive views of functional diversity and evolutionary relationships. Intriguing insights can be gained: promiscuous activities are prevalent and many divergent proteins retain "functional connectivity" via enzyme promiscuity1.Interested in the varied biological and biotechnological roles of FMN-dependent “nitroreductase” enzymes (NTRs), we undertook extensive computational and functional analyses to determine sequence, structural and functional relationships2. This large and diverse superfamily contains >80,000 sequences from all domains of life, 54 structures, and >10 enzymatic functions. Our results suggest an evolutionary model in which contemporary subgroups of the superfamily have diverged in a radial manner from a highly “evolvable” minimal flavin-binding scaffold. To investigate the diverse NTR sequence space for the capacity to catalyze nitroreduction, we synthesized >500 genes and performed high-throughput activity screening to profile 18 in vivo substrates. In vitro kinetic analysis was subsequently performed on 112 enzymes against 32 substrates (vs. 2 nicotinamide cofactors), equating to >7,000 reactions3. We demonstrated that only four of the 22 distinct superfamily subgroups display canonical nitroaromatic reductase activities. Eight additional subgroups display occasional promiscuous activities with selected substrates, and 10 subgroups display no nitroreductase activity. Structural analyses revealed the underlying molecular details: nitroreduction has emerged three distinct times in the superfamily via three unique molecular solutions - loop insertions at three different positions in the NTR scaffold, combined with the fixation of key residues, have independently led to functional specialization. These results are now facilitating the rational redesign of the NTR scaffold. Our work provides clues for functional inference for sequences of unknown function, and will aid future efforts to exploit evolvable scaffolds for engineering, and understand the emergence of functional diversity in enzyme superfamilies.

  1. Baier F, Copp JN, Tokuriki N. Biochemistry. 2016 Nov 22;55(46):6375-6388.
  2. Akiva E*, Copp JN*, Tokuriki N, Babbitt PC. Proc Natl Acad Sci U S A. 2017 114(45):E9549-E9558.
  3. Copp JN, Morales DM, Chang S, Jiang K, Akiva E, Babbitt PC, Tokuriki N. in prep.

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