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Identifying structural and dynamic changes during the Biliverdin Reductase B catalytic cycle.

Eunjeong Lee1, Matthew J McLeod2, Jasmina S Redzic1

  • 1Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Denver, Aurora, CO, United States.

Frontiers in Molecular Biosciences
|August 30, 2023
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Summary

This study explored structural and dynamic changes in Biliverdin Reductase B (BLVRB) during its catalytic cycle. Using X-ray crystallography and NMR, the researchers found that BLVRB undergoes both local and global changes. The R78-loop remains ordered through coenzyme binding and catalysis, while other regions experience dynamic exchange. These changes depend on the coenzyme’s oxidation state. The findings show that BLVRB’s function involves widespread conformational changes, not just at the active site. This suggests that BLVRB’s role in redox regulation is more complex than previously understood.

Keywords:
BLVRBNMRallosterydynamicsreductaseBiliverdin Reductase B functionEnzyme catalytic cycle dynamicsNMR in structural biologyCoenzyme binding in redox enzymes

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Area of Science:

  • Enzyme catalysis in biochemistry
  • Structural biology of redox proteins
  • Molecular dynamics in enzymatic reactions

Background:

Prior research has shown that BLVRB plays a role in cellular redox regulation. The enzyme’s structure and dynamics have been studied in limited contexts. However, the extent of structural and dynamic changes during the catalytic cycle remains unclear. No prior work had resolved whether these changes are localized or global. The role of the R78-loop in coenzyme binding was known but not fully understood. The connection between coenzyme oxidation state and enzyme dynamics had not been established. This gap motivated a detailed structural and dynamic analysis of BLVRB. The study aimed to clarify whether changes in BLVRB are confined to the active site or affect the entire enzyme.

Purpose Of The Study:

This study aimed to investigate structural and dynamic changes in BLVRB during its catalytic cycle. The researchers focused on whether these changes occur locally or globally. They sought to determine the role of the R78-loop and coenzyme binding in enzyme dynamics. The study also aimed to identify how the coenzyme’s oxidation state influences BLVRB behavior. By using X-ray crystallography and NMR, the team aimed to capture enzyme behavior at various stages. The goal was to understand how BLVRB transitions between intermediates. The study aimed to reveal whether these transitions are localized or widespread. This approach was chosen to provide a comprehensive view of BLVRB’s catalytic cycle.

Main Methods:

The researchers used X-ray crystallography to determine the apo BLVRB structure for the first time. They compared this structure to the holo form to identify global changes. Amide and Cα chemical shift perturbations were analyzed to detect environmental changes. NMR relaxation rate measurements were used to assess dynamic behavior. The study focused on the R78-loop and its interaction with the coenzyme. The team examined how coenzyme binding affects enzyme dynamics. They compared structural and dynamic changes between intermediates. This approach allowed them to track BLVRB behavior through the catalytic cycle.

Main Results:

The apo BLVRB structure showed subtle global changes compared to the holo form. A critical hydrogen bond in the R78-loop was lost in the apo structure. Coenzyme binding induced more distant global changes than substrate interactions. NMR data revealed dynamic behavior in the R78-loop during the catalytic cycle. The R78-loop remained ordered through coenzyme binding and catalysis. Other regions showed dynamic exchange between intermediates. Residue groups exhibited similar dynamic responses during the cycle. Structural and dynamic changes in BLVRB depend on coenzyme oxidation state.

Conclusions:

The study revealed that BLVRB undergoes both local and distal changes during its catalytic cycle. These changes are not confined to the active site but are allosterically coupled. The R78-loop remains ordered through coenzyme binding and catalysis. Other regions experience dynamic exchange during the cycle. The coenzyme’s oxidation state influences BLVRB’s structural and dynamic behavior. The findings suggest that BLVRB’s function involves widespread conformational changes. The study highlights the importance of considering global enzyme behavior. These conclusions align with the observed structural and dynamic data.

The apo BLVRB structure shows subtle global changes compared to the holo form, including loss of a critical hydrogen bond in the R78-loop.

NMR relaxation rate measurements were used to assess dynamic behavior, revealing that the R78-loop remains ordered while other regions experience dynamic exchange.

The R78-loop clamps the coenzyme in the active site and remains ordered through coenzyme binding and catalysis, suggesting a key role in enzyme function.

Changes in BLVRB depend on the coenzyme’s oxidation state, influencing both structural and dynamic properties during the catalytic cycle.

Coenzyme binding induces more distant global changes compared to substrate interactions, as shown by amide and Cα chemical shift perturbations.

The findings suggest that BLVRB’s redox regulation involves widespread conformational changes, not just at the active site.