2014
DOI: 10.1016/j.jmb.2013.10.012
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Exploration of Alternate Catalytic Mechanisms and Optimization Strategies for Retroaldolase Design

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 … Show more

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Cited by 36 publications
(44 citation statements)
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References 43 publications
(74 reference statements)
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“…Thus, it can be incorporated into cells to assess proteostasis network influences on RA folding without concerns about loss-or gain-of-function cellular phenotypes. The RA of focus, RA114.3 (14), adopts an eight-stranded α/β barrel fold (Fig. S1A), a common natural enzyme scaffold (15).…”
Section: Resultsmentioning
confidence: 99%
“…Thus, it can be incorporated into cells to assess proteostasis network influences on RA folding without concerns about loss-or gain-of-function cellular phenotypes. The RA of focus, RA114.3 (14), adopts an eight-stranded α/β barrel fold (Fig. S1A), a common natural enzyme scaffold (15).…”
Section: Resultsmentioning
confidence: 99%
“…[98,95] Cassette mutagenesis of active-site residues, followed by multiple rounds of error-prone polymerase chain reaction (PCR) mutagenesis and gene shuffling, afforded >1000-fold enhancements of the starting activities. [100] The most evolved retro-aldolases exhibited rate accelerations >10 8 -fold over the simple lysine-catalyzed reaction and, notably, had k cat values that are similar to or even exceed those of the best aldolase antibodies. Furthermore, their k cat /K M values approached 10 3 M -1 • s -1 , which is within a factor of 100 of the k cat /K M values for natural aldolases.…”
Section: 1123mentioning
confidence: 94%
“…Retro‐aldolases that exploit amine catalysis are the mechanistically most complex enzymes designed to date 2b. 3a, 4 They utilize a reactive active‐site lysine to promote a multistep reaction sequence involving several enzyme‐bound Schiff base intermediates. Following laboratory evolution, activities approaching those of natural class I aldolases have been attained 3b.…”
Section: Methodsmentioning
confidence: 99%