Structural Analysis of a Stereochemical Modification of Flavin Adenine Dinucleotide in Alcohol Oxidase from Methylotrophic Yeasts

Alcohol oxidase (MOX), a major peroxisomal protein of methanol-utilizing yeasts, contains two different forms of flavin adenine dinucleotide, one of which is identical with natural FAD whereas the other (mFAD) is a stereochemical modification of the natural coenzyme. This modification occurs spontaneously with FAD (but not FADH) bound to alcohol oxidase. mFAD was degraded with diphosphatase to provide authentic AMP and mFMN. The latter was degraded further with phosphatase to m-riboflavin. Analysis by H-1 and C-13 NMR spectroscopy of mFAD revealed that the isoalloxazine and adenine rings were intact and not modified structurally. However, significant differences were observed in the proton spectra in the sugar chains attached to the isoalloxazine ring (ribitol in the case of FAD). Similar observations were made for mFMN and m-riboflavin. Most striking in COSY spectra is the virtual absence of coupling between protons 2' and 3' in the sugar chain attached to the isoalloxazine ring, whereas this coupling is strong in the natural materials. Moreover, the nature of the coupling of proton 2' to protons 1a' and 1b' of the sugar chain is different in modified material. All these observations are consistent with the hypothesis that in modified cofactor the absolute configuration of carbon 2' of the sugar chain attached to the isoalloxazine ring has changed from R to S. This indicates the presence of an arabityl sugar chain rather than the ribitol present in natural FAD. A possible mechanism for this conversion is suggested.