Directed evolution of <i>Anabaena variabilis</i> phenylalanine ammonia-lyase (PAL) identifies mutants with enhanced activities

Mays, Zachary J. S.; Mohan, Karishma; Trivedi, Vikas D.; Chappell, Todd C.; Nair, Nikhil U.

doi:10.1039/d0cc00783h

Cited by 26 publications

(42 citation statements)

References 29 publications

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“…Generally, directed evolution can identify mutational hotspots using large mutant libraries, independent of their proximity to the active site. But there have only been two such studies with PALsone study resulted in modest improvement in activity 16 whereas the other, conducted by us, identified only residues within the active site 17 . Because both studies involved characterization of a limited subset of mutants following directed evolution, conclusions could only be drawn regarding very specific mutations at a few positions.…”

Section: Introductionmentioning

confidence: 93%

“…Previously, we developed a growth-coupled enrichment for rapid screening of high-activity variants of AvPAL* (the double mutant C503S-C565S PAL from Anabaena variabilis, currently used to formulate the PKU drug Pegvaliase®) in E. coli 17 . After a single round of directed evolution using this growth-coupled enrichment, we identified active site mutations G218S and M222L that possessed ~1.8-fold improvement in turnover frequency (kcat).…”

Section: Introductionmentioning

confidence: 99%

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In-depth sequence-function characterization reveals multiple paths to enhance phenylalanine ammonia-lyase (PAL) activity

Trivedi

Nb³

et al. 2021

Preprint

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Phenylalanine ammonia-lyases (PALs) deaminate L-phenylalanine to trans-cinnamic acid and ammonium and have widespread application in chemo-enzymatic synthesis, agriculture, and medicine. In particular, the PAL from Anabaena variabilis has garnered significant attention as the active ingredient in Pegvaliase®, the only FDA-approved drug treating classical phenylketonuria (PKU). Although an extensive body of literature exists on structure, substrate-specificity, and catalytic mechanism, protein-wide sequence determinants of function remain unknown, which limits the ability to rationally engineer these enzymes. Previously, we developed a high-throughput screen (HTS) for PAL, and here, we leverage it to create a detailed sequence-function landscape of PAL by performing deep mutational scanning (DMS). Our method revealed 79 hotspots that affected a positive change in enzyme fitness, many of which have not been reported previously. Using fitness values and structure-function analysis, we picked a subset of residues for comprehensive single- and multi-site saturation mutagenesis to improve the catalytic activity of PAL and identified combinations of mutations that led to improvement in reaction kinetics in cell-free and cellular contexts. To understand the mechanistic role of the most beneficial mutations, we performed QM/MM and MD and observed that different mutants confer improved catalytic activity via different mechanisms, including stabilizing first transition and intermediate states and improving substrate diffusion into the active site, and decreased product inhibition. Thus, this work provides a comprehensive sequence-function relationship for PAL, identifies positions that improve PAL activity when mutated and assess their mechanisms of action.

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Section: Introductionmentioning

confidence: 93%

Section: Introductionmentioning

confidence: 99%

In-depth sequence-function characterization reveals multiple paths to enhance phenylalanine ammonia-lyase (PAL) activity

Trivedi

Nb³

et al. 2021

Preprint

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“…From the perspective of the pathogenetic links underlying hyperphenylalaninemia development, several forms of this pathology are distinguished. A classic PKU caused by several mutations in the phenylalanine hydroxylase gene, the general result of which is a deficiency of the enzyme activity [ 17 , 19 , 20 ], and PKU forms, previously called atypical, which are associated with impaired metabolism of tetrahydrobiopterin (BH 4 ), a coenzyme involved in the hydroxylation of several amino acids, including phenylalanine [ 21 , 22 ].…”

Section: Discussionmentioning

confidence: 99%

Study of the l-Phenylalanine Ammonia-Lyase Penetration Kinetics and the Efficacy of Phenylalanine Catabolism Correction Using In Vitro Model Systems

et al. 2021

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The kinetics of L-phenylalanine ammonia-lyase (PAL) penetration into the monolayer of liver cells after its release from capsules was studied. The studies showed the absence of the effect of the capsule shell based on plant hydrocolloids on the absorption of L-phenylalanine ammonia-lyase in systems simulating the liver surface. After 120 min of incubation, in all variants of the experiment, from 87.0 to 96.8% of the enzyme penetrates the monolayer of liver cells. The combined analysis of the results concludes that the developed encapsulated form of L-phenylalanine ammonia-lyase is characterized by high efficiency in correcting the disturbed catabolism of phenylalanine in phenylketonuria, which is confirmed by the results of experiments carried out on in vitro model systems. PAL is approved for the treatment of adult patients with phenylketonuria. The encapsulated L-phenylalanine ammonia-lyase form can find therapeutic application in the phenylketonuria treatment after additional in vitro and in vivo studies, in particular, the study of preparation safety indicators. Furthermore, it demonstrated high efficacy in tumor regression and the treatment of tyrosine-related metabolic disorders such as tyrosinemia. Several therapeutically valuable metabolites biosynthesized by PAL via its catalytic action are included in food supplements, antimicrobial peptides, drugs, amino acids, and their derivatives. PAL, with improved pharmacodynamic and pharmacokinetic properties, is a highly effective medical drug.

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“…Phenylalanine ammonia lyase (PAL, EC 4.3.1.5) is the first enzyme of the general phenylpropanoid pathway catalyzing ammonia elimination from phenylalanine (Phe) to give trans -cinnamic acid, or tyrosine (Tyr) deamination to form p -coumaric acid ( p -hydroxycinnamic acids), indicating its additional tyrosine ammonia lyase (TAL) activity ( Figure 1 ) (Jun et al, 2018 ). It plays an important role in the synthesis of secondary metabolites with high biological value and has been of great interest in the food industry, agriculture, and medicine (Wang et al, 2016 ; Jun et al, 2018 ; Levy et al, 2018 ; Lin et al, 2018 ; Otto et al, 2019 ; Mays et al, 2020 ). Initially discovered as a plant enzyme, PAL has also been subsequently found in some microorganisms (Ogata et al, 1967 ; Barron et al, 2017 ; Levy et al, 2018 ).…”

Section: Introductionmentioning

confidence: 99%

“…To increase enzymatic activity, the cloning and heterogeneous expression of Rhodotorula PAL in recombinant Escherichia coli have typically been used (Cui et al, 2008 ; Jia et al, 2008 ; Babich et al, 2013 ; Zhu et al, 2013 ; Vargas-Tah et al, 2015 ; Rowles et al, 2016 ; Levy et al, 2018 ). Some methods, such as induction by the addition of amino acids, organic solvents, and surfactants (Cui et al, 2008 ), directed evolution by site-specific mutagenesis (Rowles et al, 2016 ; Mays et al, 2020 ), and coexpression of 3-deoxy- d - arabino -heptulosonate-7-phosphate synthase and transketolase (Vargas-Tah et al, 2015 ), have been further used to enhance recombinant PAL production. Here, we cloned the full-length R. glutinis PAL ( Rg PAL) gene and provided an efficient expression of the recombinant enzyme in E. coli by codon optimization.…”

Section: Introductionmentioning

confidence: 99%

Codon-Optimized Rhodotorula glutinis PAL Expressed in Escherichia coli With Enhanced Activities

Xue

Liu

et al. 2021

Front. Bioeng. Biotechnol.

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PAL (phenylalanine ammonia lyase) is important for secondary metabolite production in plants and microorganisms. There is broad interest in engineering PAL for its biocatalytic applications in industry, agriculture, and medicine. The production of quantities of high-activity enzymes has been explored by gene cloning and heterogeneous expression of the corresponding protein. Here, we cloned the cDNA of Rhodotorula glutinis PAL (RgPAL) and introduced codon optimization to improve protein expression in Escherichia coli and enzyme activities in vitro. The RgPAL gene was cloned by reverse transcription and named pal-wt. It had a full-length of 2,121 bp and encoded a 706-amino-acid protein. The pal-wt was inefficiently expressed in E. coli, even when the expression host and physical conditions were optimized. Therefore, codon optimization was used to obtain the corresponding gene sequence, named pal-opt, in order to encode the same amino acid for the RgPAL protein. The recombinant protein encoded by pal-opt, named PAL-opt, was successfully expressed in E. coli and then purified to detect its enzymatic activity in vitro. Consequently, 55.33 ± 0.88 mg/L of PAL-opt protein with a specific activity of 1,219 ± 147 U/mg and Km value of 609 μM for substrate L-phenylalanine was easily obtained. The enzyme protein also displayed tyrosine ammonia lyase (TAL)–specific activity of 80 ± 2 U/mg and Km value of 13.3 μM for substrate L-tyrosine. The bifunctional enzyme RgPAL/TAL (PAL-opt) and its easy expression advantage will provide an important basis for further applications.

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Directed evolution of Anabaena variabilis phenylalanine ammonia-lyase (PAL) identifies mutants with enhanced activities

Cited by 26 publications

References 29 publications

In-depth sequence-function characterization reveals multiple paths to enhance phenylalanine ammonia-lyase (PAL) activity

In-depth sequence-function characterization reveals multiple paths to enhance phenylalanine ammonia-lyase (PAL) activity

Study of the l-Phenylalanine Ammonia-Lyase Penetration Kinetics and the Efficacy of Phenylalanine Catabolism Correction Using In Vitro Model Systems

Codon-Optimized Rhodotorula glutinis PAL Expressed in Escherichia coli With Enhanced Activities

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