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.