Conversion of methylamine dehydrogenase to a long-chain amine dehydrogenase by mutagenesis of a single residue

Methylamine dehydrogenase (MADH) is a tryptophan tryptophylquinone (TTQ) de pendent enzyme that catalyzes the oxidative deamination of primary amines. Amino acid residues of both the TTQ-bearing beta subunit and the noncatalyt ic alpha subunit line a substrate channel that leads from the protein surfa ce to the enzyme active site. Phe55 of the alpha subunit is located at the opening of the active site. Conversion of alpha Phe55 to alanine dramatical ly alters the substrate preference of MADH. The K-m for methylamine increas es from 9 mu M to 15 mM. The preferred substrates are now primary amines wi th chain lengths of at least seven carbons. The K-m for 1,10-diaminodecane is 11 mu M, compared to 1.2 mM for wild-type MADH. Despite the large variat ion in K-m values, k(cat) values are relatively unaffected by the mutation. Molecular modeling of substrates into the crystal structure of the enzyme active site and substrate channel provides an explanation for the dramatic changes in substrate specificity caused by this mutation of a single amino acid residue.