Substrate and Inhibitor Specificities for Human Monoamine Oxidase A and B Are Influenced by a Single Amino Acid

Rani Maurice Geha,Igor Rebrin,Kevin Chen,Jean Chen Shih

doi:10.1074/jbc.m006972200

Abstract

Monoamine oxidase (MAO) is responsible for the oxidation of biogenic and dietary amines. It exists as two isoforms, A and B, which have a 70% amino acid identity and different substrate and inhibitor specificities. This study reports the identification of residues responsible for conferring this specificity in human MAO A and B. Using site-directed mutagenesis we reciprocally interchanged three pairs of corresponding nonconserved amino acids within the central portion of human MAO. Mutant MAO A-I335Y became like MAO B, which exhibits a higher preference for beta-phenylethylamine than for the MAO A preferred substrate serotonin (5-hydroxytryptamine), and became more sensitive to deprenyl (MAO B-specific inhibitor) than to clorgyline (MAO A-specific inhibitor). The reciprocal mutant MAO B-Y326I exhibited an increased preference for 5-hydroxytryptamine, a decreased preference for beta-phenylethylamine, and, similar to MAO A, was more sensitive to clorgyline than to deprenyl. These mutants also showed a distinct shift in sensitivity for the MAO A- and B-selective inhibitors Ro 41-1049 and Ro 16-6491. Mutant pair MAO A-T245I and MAO B-I236T and mutant pair MAO A-D328G and MAO B-G319D reduced catalytic activity but did not alter specificity. Our results indicate that Ile-335 in MAO A and Tyr-326 in MAO B play a critical role in determining substrate and inhibitor specificities in human MAO A and B.

Highlights

Monoamine oxidase (MAO) is responsible for the oxidation of biogenic and dietary amines
Mutant MAO A-I335Y became like MAO B, which exhibits a higher preference for ␤-phenylethylamine than for the MAO A preferred substrate serotonin (5-hydroxytryptamine), and became more sensitive to deprenyl (MAO B-specific inhibitor) than to clorgyline (MAO A-specific inhibitor)
Using site-directed mutagenesis, we constructed six MAO mutants by reciprocally interchanging three corresponding amino acid pairs in human MAO A and B within a region thought to be important for conferring substrate and inhibitor specificities

Summary

EXPERIMENTAL PROCEDURES

Materials—The QuikChange௢ site-directed mutagenesis kit was purchased from Stratagene (La Jolla, CA). Recombinant MAO A and B-encoding virus was produced by homologous recombination and cotransfection of Sf21 insect cells with the transfer vector and the linearized baculovirus DNA via the calcium phosphate method [24]. The densities of the MAOs and the bovine serum albumin bands were quantitated using the EagleSight software, and the MAO concentrations were calculated according to the linear bovine serum albumin standard. The kcat and Km values were determined as described under “Experimental Procedures” for the MAO A-preferring substrate 5-HT and the MAO B-preferring substrate PEA. The IC50 values for the irreversible inhibitors clorgyline and deprenyl were determined by preincubating the inhibitor with the homogenate for 30 min at 37 °C and assaying for the remaining activity as described [26]. Inhibitor concentrations ranged from 10Ϫ4 to 10Ϫ11 M, and the IC50 values were determined by Hill analysis

RESULTS

The substrate and inhibitor specificities exhibited by

DISCUSSION