Intracellular complexities of acquiring a new enzymatic function revealed by mass-randomisation of active-site residues.

ABSTRACT: Selection for a promiscuous enzyme activity provides substantial opportunity for competition between endogenous and newly-encountered substrates to influence the evolutionary trajectory, an aspect that is often overlooked in laboratory directed evolution studies. We selected the Escherichia coli nitro/quinone reductase NfsA for chloramphenicol detoxification by simultaneously randomising eight active-site residues and interrogating ~250,000,000 reconfigured variants. Analysis of every possible intermediate of the two best chloramphenicol reductases revealed complex epistatic interactions. In both cases, improved chloramphenicol detoxification was only observed after an R225 substitution that largely eliminated activity with endogenous quinones. Error-prone PCR mutagenesis reinforced

SUBMITTER: Hall KR 

PROVIDER: S-EPMC7738182 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature