Engineering protonation conformation of l‐aspartate‐α‐decarboxylase to relieve mechanism‐based inactivation

Abstract

Mechanism‐based inactivation of l‐aspartate‐α‐decarboxylase (PanD), which leads to irreversible modification of active site, is a major challenge in the efficient production of β‐alanine from L‐aspartic acid. In this study, a semi‐rational strategy that combined conformational dynamics and structural alignment was applied to increase the catalytic stability of Bacillus subtilis PanD (BsPanD). Using site‐saturation and C‐terminal deletion, the variant Q5 (BsPanDI46V/I88M/K104S/I126*) was generated. The catalytic half‐life and the total turnover number (TTN) of Q5 were 3.48‐fold and 2.52‐fold higher, respectively, compared with that of the parent Q0. The reasons for the differences were the prolonged distance d1 between the phenolic group of Tyr58 and pyruvoyl group of Ser25 (4.9 Å in Q0 vs. 5.5 Å in Q5), an increased difficulty for incorrect protonation to occur, and the decreased flexibility of residues in regions A, B, and C, thereby enhancing the probability of correct protonation. Variant Q5, coupled with l‐aspartase (AspA) in a 15‐L bioreactor, generated a linear cascade system using fumaric acid as a substrate, yielding 118.6 g/L β‐alanine with a product/catalyst (P/C) ratio of 5.9 g/g and a conversion > 99%. These results showed that reshaping the protonation conformation of PanD can efficiently relieve mechanism‐based inactivation and boost catalytic stability.

Enhanced catalytic efficiency and universality of L-amino acid deaminase achieved by a shorter proton transfer distance

“…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.…”

Characterization and rational modification of aspartate 4‐decarboxylase from Acinetobacter radioresistens for the production of l‐alanine

“…Recently, multienzyme cascades have been developed to synthesize high-value chemicals (France et al, 2017;Qian et al, 2020). AspA from E. coli has been employed in dual-enzyme cascades due to its superiority (Liu, Yu, et al, 2019;Qian et al, 2018).…”

The Moderately (D)efficient Enzyme: Catalysis-Related Damage In Vivo and Its Repair

“…11,18 This side reaction arises from incorrect positioning of the proton donor Tyr58. A small set of mutations that increase the difficulty of incorrect protonation substantially increased the total turnover number (from 1.2 × 10 4 to 3 × 10 4 ) without much altering k cat /K M , 11 showing again that damage hardening is evolvable.…”

“…While little can usually be done to replace damageable catalytic residues or to otherwise harden them against damage, there may be much more scope for replacing damageable bystander residues in the active site, as we have illustrated. 7,11,22…”

“…The idea that catalysis-inflicted damage is widespread and kills enzymes in vivo is not wholly without support, though. First, there are clear cases from diverse enzymes in three kingdoms of life, e.g., THI4 thiazole synthases, , aspartate α-decarboxylase, and pyruvate-formate lyase (on which we present more below). Second, the metabolic flux carried by an enzyme in vivo , k app , is generally substantially less than the enzyme’s k cat value measured in vitro , ,, meaning that enzymes apparently operate well below maximal capacity.…”