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The effect of the hydrophobic environment on the retro-aldol reaction: comparison to a computationally-designed enzyme.

ABSTRACT: Recent work on a computationally-designed retroaldolase RA-61 suggested that most of the rate-acceleration brought about by this enzyme was due to non-specific interactions with the aromatic substrate. To provide a benchmark for the role of non-specific interactions in this system, we measured the second-order rate constant for the amine-catalysed retro-aldol reaction of methodol in the presence of non-specific hydrophobic pockets such as micelles. We found that a simple micellar system, that consists of a positively-charged surfactant and a long-chain amine, can accelerate the retro-aldol reaction of methodol by 9500-fold. This effect rivals the 10(5)-fold rate acceleration of RA-61. Similar results were obtained with BSA used as the catalyst, implying that the retro-aldol reaction of methodol can be greatly accelerated by non-specific hydrophobic pockets that contain an amino group.

SUBMITTER: Schmidt J

PROVIDER: S-EPMC3919508 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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The effect of the hydrophobic environment on the retro-aldol reaction: comparison to a computationally-designed enzyme.

Schmidt Joshua J Ehasz Clayton C Epperson Michael M Klas Kimberly K Wyatt Justin J Hennig Mirko M Forconi Marcello M

Organic & biomolecular chemistry 20131105 48

Recent work on a computationally-designed retroaldolase RA-61 suggested that most of the rate-acceleration brought about by this enzyme was due to non-specific interactions with the aromatic substrate. To provide a benchmark for the role of non-specific interactions in this system, we measured the second-order rate constant for the amine-catalysed retro-aldol reaction of methodol in the presence of non-specific hydrophobic pockets such as micelles. We found that a simple micellar system, that co ...[more]

PMID: 24189834

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Project description:The evolution of the bacterial phosphotransferase system (PTS) linked to glycolysis is dependent on the availability of naturally occurring sugars. Although bacteria exhibit sugar specificities based on carbon catabolite repression, the acquisition and evolvability of the cellular sugar preference under conditions that are suboptimal for growth (e.g., environments rich in a rare sugar) are poorly understood. Here, we generated Escherichia coli mutants via a retro-aldol reaction to obtain progeny that can utilize the rare sugar d-tagatose. We detected a minimal set of adaptive mutations in the d-fructose-specific PTS to render E. coli capable of d-tagatose utilization. These E. coli mutant strains lost the tight regulation of both the d-fructose and N-acetyl-galactosamine PTS following deletions in the binding site of the catabolite repressor/activator protein (Cra) upstream from the fruBKA operon and in the agaR gene, encoding the N-acetylgalactosamine (GalNAc) repressor, respectively. Acquired d-tagatose catabolic pathways then underwent fine-tuned adaptation via an additional mutation in 1-phosphofructose kinase to adjust metabolic fluxes. We determined the evolutionary trajectory at the molecular level, providing insights into the mechanism by which enteric bacteria evolved a substrate preference for the rare sugar d-tagatose. Furthermore, the engineered E. coli mutant strain could serve as an in vivo high-throughput screening platform for engineering non-phosphosugar isomerases to produce rare sugars. IMPORTANCE Microorganisms generate energy through glycolysis, which might have preceded a rapid burst of evolution, including the evolution of cellular respiration in the primordial biosphere. However, little is known about the evolvability of cellular sugar preferences. Here, we generated Escherichia coli mutants via a retro-aldol reaction to obtain progeny that can utilize the rare sugar d-tagatose. Consequently, we identified mutational hot spots and determined the evolutionary trajectory at the molecular level. This provided insights into the mechanism by which enteric bacteria evolved substrate preferences for various sugars, accounting for the widespread occurrence of these taxa. Furthermore, the adaptive laboratory evolution-induced cellular chassis could serve as an in vivo high-throughput screening platform for engineering tailor-made non-phosphorylated sugar isomerases to produce low-calorigenic rare sugars showing antidiabetic, antihyperglycemic, and antitumor activities.

Dataset Information

The effect of the hydrophobic environment on the retro-aldol reaction: comparison to a computationally-designed enzyme.

Publications

The effect of the hydrophobic environment on the retro-aldol reaction: comparison to a computationally-designed enzyme.

Similar Datasets

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