Structural Characterization of Mutations at the Oxygen Activation Site in Monomeric Sarcosine Oxidase

Oxygen reduction and sarcosine oxidation in monomeric sarcosine oxidase (MSOX) occur at separate sites above the si- and re-faces, respectively, of the flavin ring. Mutagenesis studies implicate Lys265 as the oxygen activation site. Substitution of Lys265 with a neutral (Met, Gln, or Ala) or basic (...

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Bibliographic Details
Published in:Biochemistry (Easton) 2010-05, Vol.49 (17), p.3631-3639
Main Authors: Jorns, Marilyn Schuman, Chen, Zhi-wei, Mathews, F. Scott
Format: Article
Language:English
Subjects:
Amino Acid Substitution
BASIC BIOLOGICAL SCIENCES
Binding Sites
Catalysis
Catalytic Domain
Crystallography, X-Ray
Flavins - metabolism
GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE
ISOALLOXAZINES
KINETICS
Lysine - chemistry
Lysine - genetics
Lysine - metabolism
Models, Molecular
Molecular Structure
MORPHOLOGY
MUTAGENESIS
Mutagenesis, Site-Directed
Mutation - genetics
MUTATIONS
OXIDASES
OXIDATION
Oxidation-Reduction
Oxygen - chemistry
Oxygen - metabolism
Protein Conformation
REACTION INTERMEDIATES
REACTION KINETICS
SARCOSINE
Sarcosine - chemistry
Sarcosine - metabolism
Sarcosine Oxidase - chemistry
Sarcosine Oxidase - genetics
Sarcosine Oxidase - metabolism
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Summary:Oxygen reduction and sarcosine oxidation in monomeric sarcosine oxidase (MSOX) occur at separate sites above the si- and re-faces, respectively, of the flavin ring. Mutagenesis studies implicate Lys265 as the oxygen activation site. Substitution of Lys265 with a neutral (Met, Gln, or Ala) or basic (Arg) residue results in an ∼104- or 250-fold decrease, respectively, in the reaction rate. The overall structure of MSOX and residue conformation in the sarcosine binding cavity are unaffected by replacement of Lys265 with Met or Arg. The side chain of Met265 exhibits the same configuration in each molecule of Lys265Met crystals and is nearly congruent with Lys265 in wild-type MSOX. The side chain of Arg265 is, however, dramatically shifted (∼4−5 Å) compared with Lys265, points in the opposite direction, and exhibits significant conformational variability between molecules of the same crystal. The major species in solutions of Lys265Arg is likely to contain a “flipped-out” Arg265 and exhibit negligible oxygen activation, similar to Lys265Met. The 400-fold higher oxygen reactivity observed with Lys265Arg is attributed to a minor (
ISSN:0006-2960
1520-4995
DOI:10.1021/bi100160j