Alcohol oxidase, a major peroxisomal protein of methanol-utilizing yeasts, may possess two different forms of flavin adenine dinucleotide, classical FAD and so-called modified FAD (mFAD). Conversion of FAD into mFAD was observed both in purified preparations of the enzyme and in cells grown in batch and continuous culture. The relative amount of mFAD in the enzyme varied from 5 to 95 %, depending on the growth or storage conditions. The presence of mFAD led to a slight decrease in Vmax and a significant (about one order) decrease in the Km of alcohol oxidase with respect to methanol. The kinetics of modification measured in purified preparations of the enzyme obeyed first-order kinetics (k=0·78 h−1). The modification process was strongly inhibited by methanol, formaldehyde or hydroxylamine. Modification observed in continuous culture under steady state conditions depended on the dilution rate and could also be described as a spontaneous first-order reaction (kapp = 0·27 h−1). FAD modification could only be detected in alcohol oxidase and not in other yeast peroxisomal flavoenzymes, such as d-amino acid oxidase from Candida boidinii.
BrookeA. G.,
DijkhuizenL.,
HarderW.1986; Regulation of flavin biosynthesis in the methylotrophic yeast Hansenula polymorpha. Archives of Microbiology 145:62–70
BystrykhL. V.,
KelloggR. M.,
KruizingaW.,
DijkhuizenL.,
HarderW.,
VervoortJ.,
van BerkelW. J. H.1991; Structural and kinetic analysis of flavin adenine dinucleotide modification in alcohol oxidase from methylotrophic yeasts. In Flavins and Flavoproteins 1990Proceedings of the 10th International Symposium197–200CurtiB,
RonchiS.,
ZanettiG.
Berlin: Walter de Gruyter;
EgliTh.,
LindleyN.,
QuayleJ. R.1983; Regulation of enzyme synthesis and variation of residual concentration during carbon-limited growth of Kloeckera sp. 2201 on mixtures of methanol and glucose. Journal of General Microbiology 129:1269–1281
EgliTh.,
BosshardC.,
HamerG.1986; Simultaneous utilization of methanol-glucose mixtures by Hansenula polymorpha in a chemostat: influence of dilution rate and mixture composition on utilization pattern. Biotechnology and Bioengineering 28:1735–1741
GeisslerJ.,
GhislaS.,
KroneckP. H.1986; Flavin-dependent alcohol oxidase from yeast. Studies on the catalytic mechanism and inactivation during turnover. European Journal of Biochemistry 160:93–100
HopkinsT.,
MullerF.1987; Biochemistry of alcohol oxidase. In Proceedings of the 5th International Symposium on Microbial Growth on Cl compounds150–157van VerseveldH. W.,
DuineJ. A.
Dordrecht: Martinus Nijhoff;
RoggenkampR.,
JanowiczZ.,
StanikowskiB.,
HollenbergC.1984; Biosynthesis and regulation of the peroxisomal methanol oxidase from the methylotrophic yeast Hansenula polymorpha. Molecular and General Genetics 194:489–493
SherryB.,
AbelesR. H.1985; Mechanism of action of methanol oxidase, reconstitution of methanol oxidase with 5-deazaflavin, and inactivation of methanol oxidase by cyclopropanol. Biochemistry 24:2594–2605
WoodwardJ. R.1990; Biochemistry and applications of alcohol oxidase from methylotrophic yeasts. In Advances in Autotrophic Microbiology and One-Carbon Metabolism1205–238CoddG. A.,
DijkhuizenL.,
TabitaF. R.
Dordrecht: Martinus Nijhoff;