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J Mol Biol. 2014 Jan 9;426(1):256-71. doi: 10.1016/j.jmb.2013.10.012. Epub 2013 Oct 23.

Exploration of alternate catalytic mechanisms and optimization strategies for retroaldolase design.

Author information

1
Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
2
Laboratory of Organic Chemistry, ETH Zurich, 8093 Zurich, Switzerland.
3
Department of Biological Sciences, Northeast Structural Genomics Consortium, Columbia University, New York, NY 10027, USA.
4
Center for Advanced Biotechnology and Medicine, Rutgers, The State University of New Jersey, NJ 08854, USA.
5
Center for Advanced Biotechnology and Medicine, Rutgers, The State University of New Jersey, NJ 08854, USA; Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, NJ 08854, USA; Northeast Structural Genomics Consortium, 679 Hoes Lane, Piscataway, NJ 08854, USA.
6
Department of Biochemistry, University of Washington, Seattle, WA 98195, USA; Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195, USA. Electronic address: dabaker@u.washington.edu.

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.

KEYWORDS:

FACS; IDT; Integrated DNA Technologies; MD; enzyme design; enzyme optimization; fluorescence-activated cell sorting; molecular dynamics; protein engineering; retroaldolase

PMID:
24161950
PMCID:
PMC4104579
DOI:
10.1016/j.jmb.2013.10.012
[Indexed for MEDLINE]
Free PMC Article

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