title: A Microplate Format Assay for Real-Time Screening for New Aldolases that Accept Aryl-Substituted Acceptor Substrates.
abstract: Aldolases are potentially important biocatalysts for asymmetric synthesis of polyhydroxylated compounds. Fructose 6-phosphate aldolase (FSA) is of particular interest by virtue of its unusually relaxed dependency on phosphorylated substrates. FSA has been reported to be a promising catalyst of aldol addition involving aryl-substituted acceptors such as phenylacetaldehyde that can react with donor ketones such as hydroxyacetone. Improvement of the low intrinsic activity with bulky acceptor substrates of this type is of great interest but has been hampered by the lack of powerful screening protocols applicable in directed evolution strategies. Here we present a new screen allowing for direct spectrophotometric recording of retro-aldol cleavage. The assay utilizes an aldehyde reductase produced in vitro by directed evolution; it reduces the aldehyde product formed after cleavage of the aldol by FSA. The assay is suitable both for steady-state enzyme kinetics and for real-time activity screening in a 96-well format.
authors.affiliation: Department of Chemistry, BMC, Uppsala University, Box 576, 751 23, Uppsala, Sweden.
authors.forename: Huan
authors.initials: H
authors.lastname: Ma
authors.affiliation: Department of Chemistry, BMC, Uppsala University, Box 576, 751 23, Uppsala, Sweden.
authors.forename: Thilak Reddy
authors.initials: TR
authors.lastname: Enugala
authors.affiliation: Department of Chemistry, BMC, Uppsala University, Box 576, 751 23, Uppsala, Sweden. mikael.widersten@kemi.uu.se.
authors.forename: Mikael
authors.initials: M
authors.lastname: Widersten
mesh_terms: D000079:Acetaldehyde / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D000446:Aldehyde-Lyases / Q000378:metabolism*; D000447:Aldehydes / Q000737:chemistry; D055162:Biocatalysis; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000378:metabolism; D057166:High-Throughput Screening Assays; D007700:Kinetics; D013379:Substrate Specificity
chemical_list: D000447:Aldehydes; D029968:Escherichia coli Proteins; C116609:3-hydroxybutanal; D000446:Aldehyde-Lyases; C426024:fsaA protein, E coli; D000079:Acetaldehyde; C013192:phenylacetaldehyde
keywords: aldolases; dehydrogenases; directed evolution; high-throughput screening; retro-aldol