HAR07 encodes chorismate mutase of the methylotrophic yeast Hansenula polymorpha and is derepressed upon methanol utilization

The HARO7 gene of the methylotrophic, thermotolerant yeast Hansenula polgmo rpha was cloned by functional complementation. HARO7 encodes a monofunction al 280-amino-acid protein with chorismate mutase (EC 5.4.99.5) activity tha t catalyzes the conversion of chorismate to prephenate, a key step in the b iosynthesis of aromatic amino acids. The HARO7 gene product shows strong si milarities to primary sequences of known eukaryotic chorismate mutase enzym es. After homologous overexpression and purification of the 32-kDa protein, its kinetic parameters (k(cat) = 319.1 s(-1), n(H) = 1.56, [S](0.5) = 16.7 mM) as well as its allosteric regulatory properties were determined. Trypt ophan acts as heterotropic positive effector; tyrosine is a negative-acting , heterotropic feedback inhibitor of enzyme activity. The influence of temp erature on catalytic turnover and the thermal stability of the enzyme were determined and compared to features of the chorismate mutase enzyme of Sacc haromyces cerevisiae. Using the Cre-loxP recombination system, we construct ed mutant strains carrying a disrupted HARO7 gene that showed tyrosine auxo trophy and severe growth defects. The amount of the 0.9-kb HARO7 mRNA is in dependent of amino acid starvation conditions but increases twofold in the presence of methanol as the sole carbon source, implying a catabolite repre ssion system acting on HARO7 expression.