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Exploration of Alternate Catalytic Mechanisms and Optimization Strategies for Retroaldolase Design

Bjelic, Sinisa (author)
University of Washington, USA
Kipnis, Yakov (author)
University of Washington, USA
Wang, Ling (author)
University of Washington, USA
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Pianowski, Zbigniew (author)
ETH Zurich, Switzerland
Vorobiev, Sergey (author)
Columbia University, USA
Su, Min (author)
Columbia University, USA
Seetharaman, Jayaraman (author)
Columbia University, USA
Xiao, Rong (author)
The State University of New Jersey, USA
Kornhaber, Gregory (author)
The State University of New Jersey, USA ; University of Medicine and Dentistry of New Jersey, USA ; Northeast Structural Genomics Consortium, USA
Hunt, John F (author)
Columbia University, USA
Tong, Liang (author)
Columbia University, USA
Hilvert, Donald (author)
ETH Zurich, Switzerland
Baker, David (author)
University of Washington, USA
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 (creator_code:org_t)
Elsevier BV, 2014
2014
English.
In: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 426:1, s. 256-271
  • Journal article (peer-reviewed)
Abstract Subject headings
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  • 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.

Subject headings

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)
NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

Keyword

retroaldolase
enzyme design
protein engineering
enzyme optimization
Biokemi
Biochemistry

Publication and Content Type

ref (subject category)
art (subject category)

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