Improving the Stability and Activity of Arginine Decarboxylase at Alkaline pH for the Production of Agmatine

Abstract

Agmatine, involved in various modulatory actions in cellular mechanisms, is produced from arginine (Arg) by decarboxylation reaction using arginine decarboxylase (ADC, EC 4.1.1.19). The major obstacle of using wild-type Escherichia coli ADC (ADCes) in agmatine production is its sharp activity loss and instability at alkaline pH. Here, to overcome this problem, a new disulfide bond was rationally introduced in the decameric interface region of the enzyme. Among the mutants generated, W16C/D43C increased both thermostability and activity. The half-life (T1/2) of W16C/D43C at pH 8.0 and 60°C was 560 min, which was 280-fold longer than that of the wild-type, and the specific activity at pH 8.0 also increased 2.1-fold. Site-saturation mutagenesis was subsequently performed at the active site residues of ADCes using the disulfide-bond mutant (W16C/D43C) as a template. The best variant W16C/D43C/I258A displayed a 4.4-fold increase in the catalytic efficiency when compared with the wild-type. The final mutant (W16C/D43C/I258A) was successfully applied to in vitro synthesis of agmatine with an improved yield and productivity (>89.0% yield based on 100 mM of Arg within 5 h).

Characterization of an Arginine Decarboxylase from Streptococcus pneumoniae by Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry

“…ADCs in E. coli, a model organism for bacterial polyamine metabolic pathways, are annotated to be either constitutive (optimal catalytic activity at pH 5.2) or inducible (optimal pH is 8.23) [30][31][32]. To characterize the enzyme activity of SP_0166, optimal pH was determined.…”

Enhanced anti‐tumor activity of arginine decarboxylase through the incorporation of aromatic amino acids at the multimer‐forming interface

“…It was reported that the poly(ethylene glycol) (PEG) conjugation was used to improve the half-life in vivo, but the enzyme activity was lost upon PEG conjugation. [21] To improve RDC's stability, attempts were made to introduce a thiol bridge into the decamerforming interface, [22] but this significantly reduced catalytic efficiency.…”

“…It was reported that the poly(ethylene glycol) (PEG) conjugation was used to improve the half‐life in vivo, but the enzyme activity was lost upon PEG conjugation. [ 21 ] To improve RDC's stability, attempts were made to introduce a thiol bridge into the decamer‐forming interface, [ 22 ] but this significantly reduced catalytic efficiency. With the ultimate goal of developing an anti‐cancer drug, RDC variants with enhanced enzymatic activity have advantages, such as reducing the amount of RDC required for administration, potentially leading to reduced side effects and toxicity.…”