Expression of spinach glycolate oxidase in Saccharomyces cerevisiae: purification and characterization

Abstract

Glycolate oxidase from spinach has been expressed in Saccharomyces cerevisiae. The active enzyme was purified to near-homogeneity (purification factor approximately 1400-fold) by means of hydroxyapatite and anion-exchange chromatography. The purified glycolate oxidase is nonfluorescent and has absorbance peaks at 448 (epsilon = 9200 M-1 cm-1) and 346 nm in 0.1 M phosphate buffer, pH 8.3. The large bathochromic shift of the near-UV band indicates that the N(3) position is deprotonated at pH 8.3. A pH titration revealed that the pK of the N(3) is shifted from 10.3 in free flavin to 6.4 in glycolate oxidase. Glycolate oxidase is competitively inhibited by oxalate with a Kd of 0.24 mM at 4 degrees C in 0.1 M phosphate buffer, pH 8.3. Three pieces of evidence demonstrate that glycolate oxidase stabilizes a negative charge at the N(1)-C(2 = O) locus: the enzyme forms a tight sulfite complex with a Kd of 2.7 x 10(-7) M and stabilizes the anionic flavosemiquinone and the benzoquinoid form of 8-mercapto-FMN. Steady-state analysis at pH 8.3, 4 degrees C, yielded a Km = 1 x 10(-3) M for glycolate and Km = 2.1 x 10(-4) M for oxygen. The turnover number has been determined to be 20 s-1. Stopped-flow studies of the reductive (k = 25 s-1) and oxidative (k = 8.5 x 10(4) M-1 s-1) half-reactions have identified the reduction of glycolate oxidase to be the rate-limiting step.

Flavocytochrome P450 BM3 Mutant A264E Undergoes Substrate-dependent Formation of a Novel Heme Iron Ligand Set

“…Under the same experimental conditions, the amounts of products generated from palmitic acid and palmitoleic acid by the A264E mutant were ϳ30 -40% lower than those produced by wild-type P450 BM3, consistent with the differences in steady-state kinetics shown in Table I. In parallel studies of peroxide production during fatty acid turnover, there was no significant difference between the wild-type and A264E enzymes, suggesting that both enzymes couple NADPH oxidation to fatty acid oxygenation tightly, but that the A264E mutant is a much slower hydroxylase than wild-type P450 BM3 (44). Thus, despite the unusual spectral conversions produced upon binding longchain fatty acids, the A264E flavocytochrome retains the capacity to oxygenate fatty acids.…”

Role of tyrosine 129 in the active site of spinach glycolate oxidase

“…In a recent study of wild-type glycolate oxidase, it was confirmed that this enzyme exhibits all these features and can be regarded as a typical member of this class of flavoproteins (Macheroux et al, 1991). Moreover it was found that all its properties in solution are compatible with the X-ray crystal structure.…”

“…The mutant was identified by dideoxynucleotide sequencing. After cloning the mutant gene into the EcoRI restriction site of expression vector pGAO (Macheroux et al, 1991), the orientation was determined with an asymmetric EcoRV digest and the mutation was verified by dideoxy sequencing. Introduction of the Y129F mutation into the Escherichiu coli expression plasmid (pPM1, see Macheroux et al, 1992) was achieved by exchanging the NsiI-Sac1 fragment of the the wild-type glycolate oxidase gene with the fragment containing the Y129F mutation.…”

“…Enzymes were assayed as described by Macheroux et al (1991) except that a 50-pl sample volume was used instead of the 10 pl of wild-type glycolate oxidase. This was necessary since Y129F glycolate oxidase exhibited a comparatively low acitivity.…”

“…The Y129F mutant was prepared either from transformed yeast cells (Macheroux et al, 1991) or from the recently developed E. coli expression system (Macheroux et al, 1992). The mutant enzymes from both sources were identical in their spectral and kinetic properties.…”