ABSTRACT
Sulfite oxidase deficiency is a lethal genetic disease that results from defects either in the genes encoding proteins involved in molybdenum cofactor biosynthesis or in the sulfite oxidase gene itself. Several point mutations in the sulfite oxidase gene have been identified from patients suffering from this disease worldwide. Although detailed biochemical analyses have been carried out on these mutations, no structural data could be obtained because of problems in crystallizing recombinant human and rat sulfite oxidases and the failure to clone the chicken sulfite oxidase gene. We synthesized the gene for chicken sulfite oxidase de novo, working backward from the amino acid sequence of the native chicken liver enzyme by PCR amplification of a series of 72 overlapping primers. The recombinant protein displayed the characteristic absorption spectrum of sulfite oxidase and exhibited steady state and rapid kinetic parameters comparable with those of the tissue-derived enzyme. We solved the crystal structures of the wild type and the sulfite oxidase deficiency-causing R138Q (R160Q in humans) variant of recombinant chicken sulfite oxidase in the resting and sulfate-bound forms. Significant alterations in the substrate-binding pocket were detected in the structure of the mutant, and a comparison between the wild type and mutant protein revealed that the active site residue Arg-450 adopts different conformations in the presence and absence of bound sulfate. The size of the binding pocket is thereby considerably reduced, and its position relative to the cofactor is shifted, causing an increase in the distance of the sulfur atom of the bound sulfate to the molybdenum.
Subject(s)
Oxidoreductases Acting on Sulfur Group Donors/chemistry , Oxidoreductases Acting on Sulfur Group Donors/deficiency , Oxidoreductases Acting on Sulfur Group Donors/metabolism , Amino Acid Sequence , Animals , Arginine/chemistry , Base Sequence , Binding Sites , Chickens , Crystallography, X-Ray , Humans , Hydrogen-Ion Concentration , Kinetics , Mass Spectrometry , Models, Chemical , Models, Molecular , Molecular Sequence Data , Molybdenum/chemistry , Mutation , Oxidoreductases Acting on Sulfur Group Donors/genetics , Oxidoreductases Acting on Sulfur Group Donors/isolation & purification , Protein Binding , Protein Conformation , Recombinant Proteins/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Static Electricity , Substrate Specificity , Sulfates/metabolismABSTRACT
Disjoining pressure isotherms for water and n-octane films on glass obtained using a recently developed apparatus, based on the interferometric technique, are compared with other values reported in the literature. Our method, including a simple cleaning procedure, yields experimental results in excellent agreement both with other experimental results and with theoretical predictions. An error analysis was made which quantifies the uncertainty of our results and simultaneously allows the minimization of the errors.
