Characterization of a Novel Shewanella algae Arginine Decarboxylase Expressed in Escherichia coli

Pei, Xiao-Dong; Lu, Lianghua; Yue, Shi-Yang; Li, Ya; Liu, Xiaoling; Li, Fan; Wu, Ke-Jie; Wang, Cheng-Hua

doi:10.1007/s12033-021-00397-6

Cited by 2 publications

(2 citation statements)

References 35 publications

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“…However, roughly speaking, our final W16C/ D43C/I258A mutant is approximately 4.7-fold more active than the G. forsetii ADC at alkaline conditions. From another recent study, the catalytic efficiency of ADC from Shewanella algae was characterized (Pei et al, 2021). Even though the k cat (12.62 ± 0.68 s −1 ) for this variant was comparatively high, because of its high K M (14.55 ± 1.45 mM), when the overall k cat /K M value was compared to the final W16C/D43C/I258A mutant of this study it had lower catalytic efficiency.…”

Section: Discussionmentioning

confidence: 51%

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

et al. 2021

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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).

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Section: Discussionmentioning

confidence: 51%

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

et al. 2021

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“…Despite a higher Km value for arginine (11.3 mM), catalytic efficiency (~1.05 × 10 6 s −1 M −1 ) of SP_0166 (adapted from Figure 6) is comparable to that of E. coli ADC (1.1 × 10 6 s −1 M −1 ), and is higher than that of Paramecium bursaria chlorella virus-1 (3.3 × 10 4 s −1 M −1 ). Interestingly, while optimum pH of Shewanella algae ADC is 7.5 and its Km is 14.55 mM [49], like SP_0166, its catalytic efficiency of 8.67 × 10 2 s −1 M −1 is significantly lower compared to SP_0166 (1.05 × 10 6 s −1 M −1 ).…”

Section: Discussionmentioning

confidence: 97%

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

Lee,

Ayoola,

Shack

et al. 2024

Biomolecules

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Polyamines are polycations derived from amino acids that play an important role in proliferation and growth in almost all living cells. In Streptococcus pneumoniae (the pneumococcus), modulation of polyamine metabolism not only plays an important regulatory role in central metabolism, but also impacts virulence factors such as the capsule and stress responses that affect survival in the host. However, functional annotation of enzymes from the polyamine biosynthesis pathways in the pneumococcus is based predominantly on computational prediction. In this study, we cloned SP_0166, predicted to be a pyridoxal-dependent decarboxylase, from the Orn/Lys/Arg family pathway in S. pneumoniae TIGR4 and expressed and purified the recombinant protein. We performed biochemical characterization of the recombinant SP_0166 and confirmed the substrate specificity. For polyamine analysis, we developed a simultaneous quantitative method using hydrophilic interaction liquid chromatography (HILIC)-based liquid chromatography–tandem mass spectrometry (LC–MS/MS) without derivatization. SP_0166 has apparent Km, kcat, and kcat/Km values of 11.3 mM, 715,053 min−1, and 63,218 min−1 mM−1, respectively, with arginine as a substrate at pH 7.5. We carried out inhibition studies of SP_0166 enzymatic activity with arginine as a substrate using chemical inhibitors DFMO and DFMA. DFMO is an irreversible inhibitor of ornithine decarboxylase activity, while DFMA inhibits arginine decarboxylase activity. Our findings confirm that SP_0166 is inhibited by DFMA and DFMO, impacting agmatine production. The use of arginine as a substrate revealed that the synthesis of putrescine by agmatinase and N-carbamoylputrescine by agmatine deiminase were both affected and inhibited by DFMA. This study provides experimental validation that SP_0166 is an arginine decarboxylase in pneumococci.

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Characterization of a Novel Shewanella algae Arginine Decarboxylase Expressed in Escherichia coli

Cited by 2 publications

References 35 publications

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

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

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

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