ArticleJournal of molecular biology2014
Exploration of alternate catalytic mechanisms and optimization strategies for retroaldolase design.
Article in Journal of molecular biology, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 10 papers.
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Who cites it
10 citing papers in PubMed, 38 citations in OpenAlex.
- Perspective: Path Sampling Methods Applied to Enzymatic Catalysis.Journal of chemical theory and computation · 2022Review
- Design and optimization of enzymatic activity in a de novo β-barrel scaffold.Protein science : a publication of the Protein Society · 2022Article
- An Approach to the De Novo Synthesis of Life.Accounts of chemical research · 2022Article
- Highly active enzymes by automated combinatorial backbone assembly and sequence design.Nature communications · 2018Article
- Rosetta:MSF: a modular framework for multi-state computational protein design.PLoS computational biology · 2017Article
- Individual and collective contributions of chaperoning and degradation to protein homeostasis in E. coli.Cell reports · 2015Article
- Design of an allosterically regulated retroaldolase.Protein science : a publication of the Protein Society · 2015Article
- De novo-designed enzymes as small-molecule-regulated fluorescence imaging tags and fluorescent reporters.Journal of the American Chemical Society · 2014Article
- Heat-shock response transcriptional program enables high-yield and high-quality recombinant protein production in Escherichia coli.ACS chemical biology · 2014Article
- Small molecule probes to quantify the functional fraction of a specific protein in a cell with minimal folding equilibrium shifts.Proceedings of the National Academy of Sciences of the United States of America · 2014Article
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Authors and funding
13 authors at 5 institutions in 2 countries.
Funding
Abstract
Designed retroaldolases have utilized a nucleophilic lysine to promote carbon-carbon bond cleavage of β-hydroxy-ketones via a covalent Schiff base intermediate. Previous computational designs have incorporated a water molecule to facilitate formation and breakdown of the carbinolamine intermediate to give the Schiff base and to function as a general acid/base. Here we investigate an alternative active-site design in which the catalytic water molecule was replaced by the side chain of a glutamic acid. Five out of seven designs expressed solubly and exhibited catalytic efficiencies similar to previously designed retroaldolases for the conversion of 4-hydroxy-4-(6-methoxy-2-naphthyl)-2-butanone to 6-methoxy-2-naphthaldehyde and acetone. After one round of site-directed saturation mutagenesis, improved variants of the two best designs, RA114 and RA117, exhibited among the highest kcat (>10(-3)s(-1)) and kcat/KM (11-25M(-1)s(-1)) values observed for retroaldolase designs prior to comprehensive directed evolution. In both cases, the >10(5)-fold rate accelerations that were achieved are within 1-3 orders of magnitude of the rate enhancements reported for the best catalysts for related reactions, including catalytic antibodies (kcat/kuncat=10(6) to 10(8)) and an extensively evolved computational design (kcat/kuncat>10(7)). The catalytic sites, revealed by X-ray structures of optimized versions of the two active designs, are in close agreement with the design models except for the catalytic lysine in RA114. We further improved the variants by computational remodeling of the loops and yeast display selection for reactivity of the catalytic lysine with a diketone probe, obtaining an additional order of magnitude enhancement in activity with both approaches.
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