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Isotope Substitution of Promiscuous Alcohol Dehydrogenase Reveals the Origin of Substrate Preference in the Transition State.

ABSTRACT: The origin of substrate preference in promiscuous enzymes was investigated by enzyme isotope labelling of the alcohol dehydrogenase from Geobacillus stearothermophilus (BsADH). At physiological temperature, protein dynamic coupling to the reaction coordinate was insignificant. However, the extent of dynamic coupling was highly substrate-dependent at lower temperatures. For benzyl alcohol, an enzyme isotope effect larger than unity was observed, whereas the enzyme isotope effect was close to unity for isopropanol. Frequency motion analysis on the transition states revealed that residues surrounding the active site undergo substantial displacement during catalysis for sterically bulky alcohols. BsADH prefers smaller substrates, which cause less protein friction along the reaction coordinate and reduced frequencies of dynamic recrossing. This hypothesis allows a prediction of the trend of enzyme isotope effects for a wide variety of substrates.

SUBMITTER: Behiry EM

PROVIDER: S-EPMC5861672 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Isotope Substitution of Promiscuous Alcohol Dehydrogenase Reveals the Origin of Substrate Preference in the Transition State.

Behiry Enas M EM Ruiz-Pernia J Javier JJ Luk Louis L Tuñón Iñaki I Moliner Vicent V Allemann Rudolf K RK

Angewandte Chemie (International ed. in English) 20180219 12

The origin of substrate preference in promiscuous enzymes was investigated by enzyme isotope labelling of the alcohol dehydrogenase from Geobacillus stearothermophilus (BsADH). At physiological temperature, protein dynamic coupling to the reaction coordinate was insignificant. However, the extent of dynamic coupling was highly substrate-dependent at lower temperatures. For benzyl alcohol, an enzyme isotope effect larger than unity was observed, whereas the enzyme isotope effect was close to unit ...[more]

PMID: 29341402

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Project description:BackgroundResearch about tumor "metabolic flexibility"-the ability of cells to toggle between preferred nutrients depending on the metabolic context-has largely focused on obesity-associated cancers. However, increasing evidence for a key role for nutrient competition in the tumor microenvironment, as well as for substrate regulation of immune function, suggests that substrate metabolism deserves reconsideration in immunogenic tumors that are not strongly associated with obesity.MethodsWe compare two murine models: immunologically cold YUMM1.7 and immunologically-hot YUMMER1.7. We utilize stable isotope and radioisotope tracer-based metabolic flux studies as well as gas and liquid chromatography-based metabolomics analyses to comprehensively probe substrate preference in YUMM1.7 and YUMMER1.7 cells, with a subset of studies on the impact of available metabolites across a panel of five additional melanoma cell lines. We analyze bulk RNA-seq data and identify increased expression of amino acid and glucose metabolism genes in YUMMER1.7. Finally, we analyze melanoma patient RNA-seq data to identify potential prognostic predictors rooted in metabolism.ResultsWe demonstrate using stable isotope tracer-based metabolic flux studies as well as gas and liquid chromatography-based metabolomics that immunologically-hot melanoma utilizes more glutamine than immunologically-cold melanoma in vivo and in vitro. Analyses of human melanoma RNA-seq data demonstrate that glutamine transporter and other anaplerotic gene expression positively correlates with lymphocyte infiltration and function.ConclusionsHere, we highlight the importance of understanding metabolism in non-obesity-associated cancers, such as melanoma. This work advances the understanding of the correlation between metabolism and immunogenicity in the tumor microenvironment and provides evidence supporting metabolic gene expression as potential prognostic factors of melanoma progression and may inform investigations of adjunctive metabolic therapy in melanoma.Trial registrationDeidentified data from The Cancer Genome Atlas were analyzed.

Dataset Information

Isotope Substitution of Promiscuous Alcohol Dehydrogenase Reveals the Origin of Substrate Preference in the Transition State.

Publications

Isotope Substitution of Promiscuous Alcohol Dehydrogenase Reveals the Origin of Substrate Preference in the Transition State.

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