title: Catalytic Promiscuity of Ancestral Esterases and Hydroxynitrile Lyases.
abstract: Catalytic promiscuity is a useful, but accidental, enzyme property, so finding catalytically promiscuous enzymes in nature is inefficient. Some ancestral enzymes were branch points in the evolution of new enzymes and are hypothesized to have been promiscuous. To test the hypothesis that ancestral enzymes were more promiscuous than their modern descendants, we reconstructed ancestral enzymes at four branch points in the divergence hydroxynitrile lyases (HNL's) from esterases ∼ 100 million years ago. Both enzyme types are α/β-hydrolase-fold enzymes and have the same catalytic triad, but differ in reaction type and mechanism. Esterases catalyze hydrolysis via an acyl enzyme intermediate, while lyases catalyze an elimination without an intermediate. Screening ancestral enzymes and their modern descendants with six esterase substrates and six lyase substrates found higher catalytic promiscuity among the ancestral enzymes (P < 0.01). Ancestral esterases were more likely to catalyze a lyase reaction than modern esterases, and the ancestral HNL was more likely to catalyze ester hydrolysis than modern HNL's. One ancestral enzyme (HNL1) along the path from esterase to hydroxynitrile lyases was especially promiscuous and catalyzed both hydrolysis and lyase reactions with many substrates. A broader screen tested mechanistically related reactions that were not selected for by evolution: decarboxylation, Michael addition, γ-lactam hydrolysis and 1,5-diketone hydrolysis. The ancestral enzymes were more promiscuous than their modern descendants (P = 0.04). Thus, these reconstructed ancestral enzymes are catalytically promiscuous, but HNL1 is especially so.
authors.affiliation: Department of Biochemistry, Molecular Biology & Biophysics and The Biotechnology Institute, University of Minnesota , 1479 Gortner Avenue, Saint Paul, Minnesota 55108, United States.
authors.forename: Titu
authors.initials: T
authors.lastname: Devamani
authors.affiliation: Department of Biochemistry, Molecular Biology & Biophysics and The Biotechnology Institute, University of Minnesota , 1479 Gortner Avenue, Saint Paul, Minnesota 55108, United States.
authors.forename: Alissa M
authors.initials: AM
authors.lastname: Rauwerdink
authors.affiliation: Department of Ecology, Evolution & Behavior and The Biotechnology Institute, University of Minnesota , 1479 Gortner Avenue, Saint Paul, Minnesota 55108, United States.
authors.forename: Mark
authors.initials: M
authors.lastname: Lunzer
authors.affiliation: Department of Biochemistry, Molecular Biology & Biophysics and The Biotechnology Institute, University of Minnesota , 1479 Gortner Avenue, Saint Paul, Minnesota 55108, United States.
authors.forename: Bryan J
authors.initials: BJ
authors.lastname: Jones
authors.affiliation: Department of Biochemistry, Molecular Biology & Biophysics and The Biotechnology Institute, University of Minnesota , 1479 Gortner Avenue, Saint Paul, Minnesota 55108, United States.
authors.forename: Joanna L
authors.initials: JL
authors.lastname: Mooney
authors.affiliation: Department of Chemistry, University of Minnesota , 220 Pleasant Street, Minneapolis, Minnesota 55455, United States.
authors.forename: Maxilmilien Alaric O
authors.initials: MA
authors.lastname: Tan
authors.affiliation: School of Biotechnology, East China University of Science, Technology , Meilong Road 130, Shanghai 200237, PR China.
authors.forename: Zhi-Jun
authors.initials: ZJ
authors.lastname: Zhang
authors.affiliation: School of Biotechnology, East China University of Science, Technology , Meilong Road 130, Shanghai 200237, PR China.
authors.forename: Jian-He
authors.initials: JH
authors.lastname: Xu
authors.affiliation: Department of Ecology, Evolution & Behavior and The Biotechnology Institute, University of Minnesota , 1479 Gortner Avenue, Saint Paul, Minnesota 55108, United States.|Institute of Ecology and Evolution, Sun Yat-sen University , No.135, Xinggang West Road, Guangzhou, 510275, PR China.
authors.forename: Antony M
authors.initials: AM
authors.lastname: Dean
authors.affiliation: Department of Biochemistry, Molecular Biology & Biophysics and The Biotechnology Institute, University of Minnesota , 1479 Gortner Avenue, Saint Paul, Minnesota 55108, United States.
authors.forename: Romas J
authors.initials: RJ
authors.lastname: Kazlauskas
mesh_terms: D000446:Aldehyde-Lyases / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis; D002264:Carboxylic Acids / Q000737:chemistry / Q000378:metabolism; D004950:Esterases / Q000737:chemistry / Q000378:metabolism*; D004952:Esters / Q000737:chemistry / Q000378:metabolism; D006856:Hydrogen Cyanide / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D009570:Nitriles / Q000737:chemistry / Q000378:metabolism
chemical_list: D002264:Carboxylic Acids; D004952:Esters; D009570:Nitriles; C430593:cyanohydrin; D006856:Hydrogen Cyanide; D004950:Esterases; D000446:Aldehyde-Lyases; C021541:mandelonitrile lyase