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 (MAOTb) is about 36 % identical to both putrescine oxidase and
human MAOs and can be efficiently produced in Escherichia coli. MAOTb preferentially acts on n-alkyl mono-
amines but shows detectable activity also on polyamines and aromatic monoamines. The crystal structures of
MAOTb 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 MAOTb 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 MAOTb 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 kcat/
KM values was found on n-alkyl-monoamines and on aromatic amines.