Structural and mechanistic insights into catalytic promiscuity of PLP ‐dependent enzymes of fold type IV : A case study of aminodeoxychorismate lyase from Micromonospora aurantiaca

Pyridoxal-5'-phosphate (PLP)-dependent enzymes of fold type IV exhibit remarkable catalytic diversity, catalyzing either S- and R-selective transamination or β-elimination within a highly conserved structural scaffold. Here, we report the identification, biochemical characterization, and structural analysis of PLP fold type IV 4-amino-4-deoxychorismate lyase (ADCL) from Micromonospora aurantiaca (Micau5708). Sequence-structural analysis revealed a hybrid architecture of the active site of Micau5708. It combines a D-amino acid transaminase (DATA) conserved phenylalanine (F30) and arginine (R87) residues with an ADCL-specific threonine residue (T32) and a (R)-selective amine transaminase-specific histidine residue (H25). While Micau5708 shows only ADCL activity, substitution H25R introduces transamination activity, preserving ADCL activity. The 1.85 Å resolution crystal structure of Micau5708 in complex with substrate analog chorismate provides the first direct visualization of substrate binding in an ADCL active site. Site-directed mutagenesis, together with comparative analysis of DATAs of group II demonstrates that ADCL/DATA catalytic promiscuity is enabled by a specific amino acid combination in the active site: phenylalanine, threonine, and arginine residues (F30, T32, and R87), and arginine at position 25 with a flexible side chain. Furthermore, the Micau5708-chorismite complex structure has helped revise the catalytic mechanism of ADCLs, demonstrating the T32 assistance in protonation of the oxygen atom rather than the β-methylene carbon of the leaving enol-pyruvate. Together, these findings provide new insights into the structural basis of reaction specificity, catalytic promiscuity, and evolutionary plasticity within the PLP fold type IV enzyme superfamily. They also highlight the limitations of sequence-based functional prediction.