Use of indirect site-directed mutagenesis to alter the substrate specificity of methylamine dehydrogenase.
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 alphaPhe(55) controls this substrate specificity. When alphaPhe(55) is replaced with Ala, this preference is reversed with alphaF55A 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 alphaPhe(55) was repositioned by site-directed mutagenesis of residue betaIle(107). This residue makes close contact with alphaPhe(55) and restricts its movement. betaI107V MADH exhibits a strong preference for propylamine, and betaI107N 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 alphaF55I 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.
Wang
Yongting
Y
Department of Biochemistry, University of Mississippi Medical Center, Jackson, Mississippi 39216-4505, USA.
Sun
Dapeng
D
Davidson
Victor L
VL
D001483:Base Sequence; D017931:DNA Primers; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D017931:DNA Primers; C021567:methylamine dehydrogenase; D000587:Oxidoreductases Acting on CH-NH Group Donors