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Related Experiment Videos

Biotin sulfoxide reductase: Tryptophan 90 is required for efficient substrate utilization.

Veronica V Pollock1, Richard C Conover, Michael K Johnson

  • 1Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa, FL 33612, USA.

Archives of Biochemistry and Biophysics
|December 31, 2002
PubMed
Summary

Investigating biotin sulfoxide reductase (BSOR), this study reveals tryptophan 90 is crucial for its catalytic activity. Mutating this residue significantly impairs enzyme function, suggesting it acts as a hydrogen bond donor to the molybdenum center.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Molybdenum-containing enzymes

Background:

  • Biotin sulfoxide reductase (BSOR) from Rhodobacter sphaeroides f. sp. denitrificans catalyzes d-biotin d-sulfoxide reduction.
  • BSOR utilizes the molybdopterin guanine dinucleotide (MGD) cofactor and shares sequence homology with dimethyl sulfoxide reductase (DMSOR).
  • A conserved active-site tryptophan (W90) in BSOR is hypothesized to hydrogen bond with the Mo(VI) oxo group.

Purpose of the Study:

  • To investigate the role of the conserved active-site tryptophan residue (W90) in BSOR catalysis.
  • To elucidate the function of W90 in substrate binding and molybdenum center redox states.

Main Methods:

  • Site-directed mutagenesis was employed to replace W90 with phenylalanine, tyrosine, and alanine.
  • Purification of wild-type and mutant BSOR proteins.

Related Experiment Videos

  • Enzyme activity assays, visible absorption, circular dichroism, and electron paramagnetic resonance (EPR) spectroscopy were utilized.

    • Main Results:

    • Mutant BSOR proteins (W90F, W90Y, W90A) were purified and contained MGD but exhibited significantly reduced catalytic activity (W90F retained 3.4% activity).
    • Mutations led to perturbed spectral properties, indicating altered molybdenum oxidation states.
    • EPR studies showed that W90 mutants trapped molybdenum in an intermediate Mo(V) state, unlike wild-type BSOR.

    Conclusions:

    • W90 is essential for BSOR catalysis, likely by acting as a hydrogen bond donor to the Mo(VI) oxo group.
    • This interaction stabilizes the active molybdenum center and facilitates the catalytic cycle.
    • The findings provide critical insights into the mechanism of molybdenum-containing hydroxylases.