DISCUSSION
In this study, an engineered α-keto acid-producing Pmi LAAD
recombinant strain with enhanced catalytic efficiency toward amino acid
substrates harboring different side chains is reported. First, combined
with the catalytic mechanism of Pmi LAAD, the θ and catalytic
distance D1 were identified as the key factor affecting catalytic
efficiency. Second, a mutational strategy to change the angle between
α-hydrogen and isoalloxazine ring and shorten D1 and increase catalytic
efficiency was proposed, leading to two improved variants (W1 and W2).
Third, strain S3 was generated by co-expressing W1 and W2 in E.
coli . This last variant exhibited high catalytic efficiency toward all
the selected amino acids. In summary, these results describe a protein
engineering strategy for improving the catalytic efficiency ofPmi LAAD or other enzymes that share a similar mechanism and
participate in α-keto acid synthesis.
Understanding the catalytic mechanism of a reaction is of great
significance while performing enzyme engineering. Existing reports on
the catalytic mechanism of LAADs have described the overall reaction
process, without explaining how protons are transferred between the
substrate and FAD(Molla et al. 2017; Rosini et al. 2017).
Here, we found that an hydride ion of substrate was required for the
transfer of αC-H to FAD N(5), and the distance D1 was identified as the
key factor affecting this step, as well as overall Pmi LAAD
catalytic efficiency. Thus, our results revealed the catalytic mechanism
of LAAD and provided effective guidance for the following protein
engineering steps.
Shortening the catalytic distance D1 improved LAAD efficiency.
Previously, the engineering of LAAD had focused on structural
alignment(Wu et al. 2020), H-bond analysis(Pei et al.2020), pocket volume, and steric hindrance(Yuan et al. 2019),
with the intent of minimizing product inhibition or maximizing catalytic
efficiency. For example, Pei et al. reduced product inhibition caused by
α-keto valine (KPI -Val) from 0.8
to 5.4 mM by changing the H-bond interaction to generated variantPmi LAADS98A/T105A/S106A/L341A(Pei et al.2020). Wu et al. generated the variant Pmi LAADF93S/P186A/M394V/F184S, which exhibited a 6.6-fold
improvement in specific activity toward L-Phe(Wu et al. 2020). In
contrast, in the present study, the protein engineering strategy was
based on analysis of the underlying catalytic mechanism and, hence, on
shortening of D1. Indeed, this strategy has proven to be very effective
at improving LAAD catalytic efficiency. In future investigations, more
biological tools, such as X-ray structures of the enzyme-substrate
complexes, can be used to further understand the relationship between
substrate specificities and the D1 distance and the mechanisms that
enhance the LAAD efficiency.
The concept of multi-enzyme cascade was used to combine W1 and W2
variants in a single, highly efficient, and universal whole-cell
catalyst. This whole-cell catalyst was constructed to be used in
industrial applications to produce α-keto acids. This multi-enzyme
cascade was necessary when an enzyme was used to efficiently catalyze
all substrates. Multi-enzyme cascades are often used in multi-step
reactions(Qian et al. 2020; Shi et al. 2018). However, in
the present study, W1 and W2, which catalyze the same deamination
reaction, were cascaded simultaneously to improve the catalytic
efficiency and substrate specificity of the same enzyme in a one-pot
strategy. As proven here, this cascade method can be used to fine-tune
the same reaction, whereby one variant is used to catalyze part of the
reaction that the other variant cannot complete. Furthermore, this
cascade method could replace conventional combination mutations, wherein
the pairing of successful single mutated residues does not produce the
expected result.
In summary, the biocatalytic process used in this study provides an
attractive strategy to facilitate the industrial production of α-keto
acids. Furthermore, the proposed approach based on shortening of the
catalytic distance and a multi-enzyme cascade reaction, offers an
attractive strategy and reference for a more widespread application of
this technique.