title: Catalysis by a de novo zinc-mediated protein interface: implications for natural enzyme evolution and rational enzyme engineering.
abstract: Here we show that a recent computationally designed zinc-mediated protein interface is serendipitously capable of catalyzing carboxyester and phosphoester hydrolysis. Although the original motivation was to design a de novo zinc-mediated protein-protein interaction (called MID1-zinc), we observed in the homodimer crystal structure a small cleft and open zinc coordination site. We investigated if the cleft and zinc site at the designed interface were sufficient for formation of a primitive active site that can perform hydrolysis. MID1-zinc hydrolyzes 4-nitrophenyl acetate with a rate acceleration of 10(5) and a k(cat)/K(M) of 630 M(-1) s(-1) and 4-nitrophenyl phosphate with a rate acceleration of 10(4) and a k(cat)/K(M) of 14 M(-1) s(-1). These rate accelerations by an unoptimized active site highlight the catalytic power of zinc and suggest that the clefts formed by protein-protein interactions are well-suited for creating enzyme active sites. This discovery has implications for protein evolution and engineering: from an evolutionary perspective, three-coordinated zinc at a homodimer interface cleft represents a simple evolutionary path to nascent enzymatic activity; from a protein engineering perspective, future efforts in de novo design of enzyme active sites may benefit from exploring clefts at protein interfaces for active site placement.
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599-7260, USA.
authors.forename: Bryan S
authors.initials: BS
authors.lastname: Der
authors.forename: David R
authors.initials: DR
authors.lastname: Edwards
authors.forename: Brian
authors.initials: B
authors.lastname: Kuhlman
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D020134:Catalytic Domain; D019143:Evolution, Molecular; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008667:Metalloproteins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D015202:Protein Engineering; D015032:Zinc / Q000378:metabolism*
chemical_list: D008667:Metalloproteins; D009596:Nitrophenols; D009943:Organophosphorus Compounds; C008644:nitrophenylphosphate; C008642:4-nitrophenyl acetate; D015032:Zinc