Use of indirect site-directed mutagenesis to alter the substrate specificity of methylamine dehydrogenase

Abstract

Methylamine dehydrogenase (MADH) is a tryptophan tryptophylquinone-dependent enzyme that catalyzes the oxidative deamination of primary amines. Native MADH exhibits a strong preference for methylamine over longer carbon chain amines. Residue αPhe55 controls this substrate specificity. When αPhe55 is replaced with Ala, this preference is reversed with αF55A MADH preferring long-chain amines with at least seven carbons (Zhu, Z., Sun, D., and Davidson, V. L. (2000) Biochemistry 39, 11184-11186). To further modulate the substrate specificity of MADH, the side-chain of αPhe55 was repositioned by site-directed mutagenesis of residue βIle107. This residue makes close contact with αPhe55 and restricts its movement. βI107V MADH exhibits a strong preference for propylamine, and βI107N MADH exhibits a preference for 1-aminopentane. Thus, it has been possible to create forms of MADH that exhibit a preference for amines with carbon chain lengths of one, three, five, or seven carbons. The ability to discriminate between amines of different chain length was essentially abolished by an αF551 mutation. Molecular modeling studies with the known crystal structure are described that provide an explanation for these results. These results provide an example of a design-based approach to protein engineering in which site-directed mutagenesis on one residue can be used to reposition another residue to specifically alter enzyme specificity.