Product release mechanism and the complete enzyme catalysis cycle in yeast cytosine deaminase (yCD): A computational study

Zhao, Yuan; She, Nai; Zhang, Xin; Wang, Chaojie; Mo, Yirong

doi:10.1016/j.bbapap.2017.05.001

Cited by 10 publications

(17 citation statements)

References 32 publications

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“…Fusion of the ODDD to the C-terminus of yCD may disrupt folding or function of the yCD for steric reasons, since the Cterminus of yCD is involved in substrate binding and product release. 13,14 This is consistent with the observation that HOPE-4, the fusion of this type with the longest linker, conferred the highest 5-FC toxicity among HOPE fusions 1−4. Accumulation of HIF-1α in HT1080 Cells in Mock-Hypoxia Using Cobalt Chloride.…”

Section: ■ Results and Discussionsupporting

confidence: 90%

Protein-Programmed Accumulation of Yeast Cytosine Deaminase in Cancer Cells in Response to Mock-Hypoxia

Warren

Patel

Eshleman³

et al. 2019

ACS Synth. Biol.

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One limitation of gene-directed enzyme prodrug therapy (GDEPT) is the difficulty in selectively and efficiently transducing cancer cells with the gene encoding a prodrug-converting enzyme. To circumvent this issue, we sought to move the selectivity from the gene delivery level to the protein level. We developed fusion proteins of the prodrug-activating enzyme yeast cytosine deaminase (yCD) and the oxygen-dependent degradation domain (ODDD) of HIF-1α, a domain that regulates the accumulation of HIF-1α in an oxygen-dependent manner. We called these HOPE fusions for HIF1-α Oxygen-dependent degradation domain/Prodrug-converting Enzyme. The HOPE fusions were designed to selectively accumulate in cells experiencing hypoxia and thus selectively cause conversion of the prodrug 5-fluorocytosine (5-FC) to the chemotherapeutic 5-fluorouracil (5-FU) where oxygen levels are low (e.g., at the center of a tumor). Consistent with our hypothesis, HT1080 fibrosarcoma cells transduced with HOPE fusion genes exhibited increased fusion protein accumulation and increased sensitization to 5-FC in mock-hypoxia.

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Section: ■ Results and Discussionsupporting

confidence: 90%

Protein-Programmed Accumulation of Yeast Cytosine Deaminase in Cancer Cells in Response to Mock-Hypoxia

Warren

Patel

Eshleman³

et al. 2019

ACS Synth. Biol.

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“…4C), and that the increase appears to primarily affect the k cat value (2.2 ± 0.10 s −1 and 1.4 ± 0.07 s −1 under UV and blue light, respectively; Table S2). These results are consistent with previous mechanistic studies which have found that product release is rate-limiting in the overall mechanism of the enzyme 13,15 , and Trp152 plays a key role in this process. Our NMR and fluorescence experiments suggest that the environment of Trp152 is altered in yCD-Opt1 by labeling and irradiation, potentially leading to the observed changes in k cat .…”

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confidence: 93%

Computational Design of a Photocontrolled Cytosine Deaminase

et al. 2017

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There is growing interest in designing spatiotemporal control over enzyme activities using noninvasive stimuli such as light. Here, we describe a structure-based, computation-guided predictive method for reversibly controlling enzyme activity using covalently attached photoresponsive azobenzene groups. Applying the method to the therapeutically useful enzyme yeast cytosine deaminase, we obtained a ∼3-fold change in enzyme activity by the photocontrolled modulation of the enzyme's active site lid structure, while fully maintaining thermostability. Multiple cycles of switching, controllable in real time, are possible. The predictiveness of the method is demonstrated by the construction of a variant that does not photoswitch as expected from computational modeling. Our design approach opens new avenues for optically controlling enzyme function. The designed photocontrolled cytosine deaminases may also aid in improving chemotherapy approaches that utilize this enzyme.

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“…Each monomer consists of a five‐strand beta sheet sandwiched between two solvent exposed α‐helices and another four α‐helices near the dimer interface (Figure S2). Important residues for activity include those involved in the catalytic step (H62, C91, C94), in substrate entry or product release (F114, W152, and I156), and in product stabilization before release (N51, D155, and E64) . The mutations identified in this study (C71A, E75P, and H127V) were not at residues directly involved in catalysis.…”

Section: Resultsmentioning

confidence: 99%

Comprehensive mutagenesis on yeast cytosine deaminase yields improvements in 5‐fluorocytosine toxicity in HT1080 cells

et al. 2019

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Improved prodrug‐activating enzymes have the potential to increase the therapeutic efficacy of gene‐directed enzyme prodrug therapy (GDEPT). Yeast cytosine deaminase (yCD) is commonly used to convert the prodrug 5‐fluorocytosine (5‐FC) to the chemotherapeutic 5‐fluorouracil for GDEPT. Mutagenesis studies on yCD aimed at improving its application in GDEPT have been limited to subsets of residues or have sought to improve a single property of the enzyme. We performed comprehensive site‐saturation mutagenesis (CSM) on yCD designed to create all 2,983 possible unique protein mutants with a single amino acid substitution. We identified active variants through Escherichia coli genetic complementation and screened these mutants, and combinations thereof, for increased ability to sensitize E. coli and HT1080 fibrosarcoma cells to 5‐FC. Several mutants identified in this study showed increased sensitization ability for both E. coli and HT1080 cells indicating that CSM is an effective directed evolution tool for identifying unexpectedly beneficial mutations.

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Product release mechanism and the complete enzyme catalysis cycle in yeast cytosine deaminase (yCD): A computational study

Cited by 10 publications

References 32 publications

Protein-Programmed Accumulation of Yeast Cytosine Deaminase in Cancer Cells in Response to Mock-Hypoxia

Protein-Programmed Accumulation of Yeast Cytosine Deaminase in Cancer Cells in Response to Mock-Hypoxia

Computational Design of a Photocontrolled Cytosine Deaminase

Comprehensive mutagenesis on yeast cytosine deaminase yields improvements in 5‐fluorocytosine toxicity in HT1080 cells

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