The catalytic mechanism of PmiLAAD
To effectively guide the protein engineering of Pmi LAAD, we relied on the hydrogen transfer mechanism of flavin-dependent enzymes(Mattevi et al. 1996; Molla et al. 2017; Mottaet al. 2016; Sandoval et al. 2021; Umhau et al.2000). According to this hypothetical mechanism, Pmi LAAD first transfers an hydride ion (one proton and 2 electrons) of the substrate, from the α-carbon (αC-H) to the cofactor FAD. As shown in Scheme 2B, the αC-H is transferred to the FAD isoalloxazine ring N(5), thus forming the anionic form of reduced FAD (FADH-); while the NH3+-H is accepted by the active site water molecule. The transfer of the hydride is accompanied by transformation of the L-amino acids to an imino acids. Next, the H-atoms are transferred from FADH- to an O-atom through cytochrome b-like proteins, thereby regenerating FAD and releasing H2O. In the meantime, the imino acids undergoes spontaneous hydrolysis to α-keto acids in aqueous solution and ammonia was released (Scheme 2A). Given that αC-H of the substrate participate in H-atom transfer from αC-H to FAD N(5), we speculated that the distance between αC-H and FAD N(5)(Molla et al. 2017; Williamset al. 2000)(called catalytic distance D1) might be the key factor affecting Pmi LAAD catalytic efficiency.
To determine how D1 affected Pmi LAAD catalytic efficiency, a homology model of Pmi LAAD was built using Proteus myxofaciens LAAD (Pma LAAD) as template (PDB ID: 5fjn)(Mottaet al. 2016) (Figure 2). The model was docked with the six selected amino acid substrates and then subjected to kinetic simulations to evaluate the various binding conformations. Accordingly, D1 ofPmi LAAD was estimated as: D1Leu (2.4 Å) < D1Met (2.8 Å) < D1Val (2.9 Å) < D1Phe (3.0 Å) < D1Arg (3.3 Å) < D1Glu (3.8 Å).(The detailed process of homology modeling and molecular docking and the method used to measure D1 can be found in Supporting Information.) The relative enzymatic activity ofPmi LAAD decreased with increasing D1: L-Leu (100%) > L-Met (73.5%) > L-Val (66.3%) > L-Phe (54.1%) > L-Arg (28.2%) > L-Glu (8.6%) (Figure 1C). The negative correlation between catalytic efficiency and D1 could be explained by a short D1 facilitating proton transfer and consequently increasing catalytic efficiency. Therefore, shortening D1 by protein engineering could potentially improve Pmi LAAD catalyticy efficiency.