Selection of a Promiscuous Minimalist cAMP Phosphodiesterase from a Library of De Novo Designed Proteins

Abstract

The ability of unevolved amino acid sequences to become biological catalysts was key to the emergence of life on Earth. However, billions of years of evolution separate complex modern enzymes from their simpler early ancestors. To study how unevolved sequences can develop new functions, we screened for enzymatic activity in a collection of > 1 million novel sequences based on a de novo 4-helix bundle library of semi-random sequences. To mirror evolutionary selection for biological function, we screened the collection using ultrahigh-throughput droplet microfluidics to identify features that yield phosphoesterase activity. Characterization of active hits demonstrated that acquiring new function required a large jump in sequence space: screening enriched for truncations that removed > 40% of the protein chain and introduced a catalytically important cysteine. The truncated protein dimerized into a dynamic -helical structure, consistent with the idea that gain of function was accompanied by an increase in structural dynamics relative to the parental 4-helix bundle. The purified protein catalyzes the hydrolysis of a range of phosphodiesters, with the greatest activity toward the biological second messenger cyclic AMP (cAMP). The novel cAMPase is a manganese-dependent metalloenzyme and catalyzes cAMP hydrolysis with a rate acceleration on the order of 109 and catalytic proficiency on the order of 1014 M-1, comparable to large enzymes shaped by billions of years of evolution. These findings suggest that fragmentation to modular primordial peptides can be a fertile avenue for introducing structural and functional diversity into proteins. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=67 SRC="FIGDIR/small/528392v2_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@180e004org.highwire.dtl.DTLVardef@1f32a75org.highwire.dtl.DTLVardef@16876aforg.highwire.dtl.DTLVardef@147ffc9_HPS_FORMAT_FIGEXP M_FIG C_FIG