title: Computational design of catalytic dyads and oxyanion holes for ester hydrolysis.
abstract: Nucleophilic catalysis is a general strategy for accelerating ester and amide hydrolysis. In natural active sites, nucleophilic elements such as catalytic dyads and triads are usually paired with oxyanion holes for substrate activation, but it is difficult to parse out the independent contributions of these elements or to understand how they emerged in the course of evolution. Here we explore the minimal requirements for esterase activity by computationally designing artificial catalysts using catalytic dyads and oxyanion holes. We found much higher success rates using designed oxyanion holes formed by backbone NH groups rather than by side chains or bridging water molecules and obtained four active designs in different scaffolds by combining this motif with a Cys-His dyad. Following active site optimization, the most active of the variants exhibited a catalytic efficiency (k(cat)/K(M)) of 400 M(-1) s(-1) for the cleavage of a p-nitrophenyl ester. Kinetic experiments indicate that the active site cysteines are rapidly acylated as programmed by design, but the subsequent slow hydrolysis of the acyl-enzyme intermediate limits overall catalytic efficiency. Moreover, the Cys-His dyads are not properly formed in crystal structures of the designed enzymes. These results highlight the challenges that computational design must overcome to achieve high levels of activity.
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.
authors.forename: Florian
authors.initials: F
authors.lastname: Richter
authors.forename: Rebecca
authors.initials: R
authors.lastname: Blomberg
authors.forename: Sagar D
authors.initials: SD
authors.lastname: Khare
authors.forename: Gert
authors.initials: G
authors.lastname: Kiss
authors.forename: Alexandre P
authors.initials: AP
authors.lastname: Kuzin
authors.forename: Adam J T
authors.initials: AJ
authors.lastname: Smith
authors.forename: Jasmine
authors.initials: J
authors.lastname: Gallaher
authors.forename: Zbigniew
authors.initials: Z
authors.lastname: Pianowski
authors.forename: Roger C
authors.initials: RC
authors.lastname: Helgeson
authors.forename: Alexej
authors.initials: A
authors.lastname: Grjasnow
authors.forename: Rong
authors.initials: R
authors.lastname: Xiao
authors.forename: Jayaraman
authors.initials: J
authors.lastname: Seetharaman
authors.forename: Min
authors.initials: M
authors.lastname: Su
authors.forename: Sergey
authors.initials: S
authors.lastname: Vorobiev
authors.forename: Scott
authors.initials: S
authors.lastname: Lew
authors.forename: Farhad
authors.initials: F
authors.lastname: Forouhar
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Kornhaber
authors.forename: John F
authors.initials: JF
authors.lastname: Hunt
authors.forename: Gaetano T
authors.initials: GT
authors.lastname: Montelione
authors.forename: Liang
authors.initials: L
authors.lastname: Tong
authors.forename: K N
authors.initials: KN
authors.lastname: Houk
authors.forename: Donald
authors.initials: D
authors.lastname: Hilvert
authors.forename: David
authors.initials: D
authors.lastname: Baker
mesh_terms: D055162:Biocatalysis; D020134:Catalytic Domain; D015195:Drug Design; D004950:Esterases / Q000737:chemistry* / Q000378:metabolism*; D004952:Esters; D006860:Hydrogen Bonding; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular
chemical_list: D004952:Esters; D004950:Esterases