Altering substrate specificity of a thermostable bacterial monoamine oxidase by structure-based mutagenesis.

Bacterial monoamine oxidases (MAOs) are FAD-dependent proteins catalyzing a relevant reaction for many industrial biocatalytic applications, ranging from production of enantiomerically pure building blocks for pharmaceutical synthesis to biosensors for monitoring food and beverage quality. The thermostable MAO enzyme from Thermoanaerobacterales bacterium (MAO) is about 36 % identical to both putrescine oxidase and human MAOs and can be efficiently produced in Escherichia coli. MAO preferentially acts on n-alkyl monoamines but shows detectable activity also on polyamines and aromatic monoamines. The crystal structures of MAO in complex with putrescine, benzylamine, spermidine and n-heptylamine at resolution ranging from 1.6 to 2.3 Å resolution revealed the binding mode of substrates to the enzyme. The MAO active site is highly conserved in the inner part of the cavity in front of the flavin ring (re face), where the presence of two tyrosine residues creates the substrate amine binding site that is found also in human MAOs. Instead, more distantly from the flavin, the entrance of the catalytic site is much more open in MAO and features a different arrangement of amino acids. Site-directed mutagenesis targeting residues Ala168, Thr199 and Val324 allowed the identification of key residues in ligand binding to alter substrate specificity. The A168D variant showed a higher activity on putrescine than wild-type, whereas by replacing either Thr199 or Val324 to Trp a marked enhancement in k/K values was found on n-alkyl-monoamines and on aromatic amines.