PAL Variant Composition for Stable Low-Aggregation PKU Therapy
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Solution Overview
Problem
Existing phenylalanine ammonia lyase (PAL) therapies for treating phenylketonuria (PKU) face challenges such as reduced specific activity, proteolytic instability, and immunogenicity, with a need for improved kinetic characteristics, longer biological half-life, and reduced immunogenicity.
Innovation Solution
Development of PAL variants from Anabaena variabilis with specific amino acid substitutions, including C503S/C565L, C503S/C565P, C503L/C565P, T102R, and others, enhancing catalytic activity, thermal stability, and reducing aggregation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If PAL is used to treat PKU, then phenylalanine levels can be reduced, but the enzyme exhibits reduced specific activity and proteolytic instability
Solution Approach 1:
The patent applies parameter changes by systematically modifying amino acid residues at specific positions (C64, C318, C503, C565, and combinations thereof) to alter the enzyme's kinetic and stability parameters. The variant PAL enzymes exhibit improved catalytic efficiency (kcat/Km) and resistance to proteolytic degradation while maintaining phenylalanine decarboxylase activity, resolving the contradiction between stability and productivity
Solution Approach 2:
The patent applies local quality by introducing specific amino acid substitutions at targeted positions within the PAL enzyme structure. These localized changes at cysteine residues and other specific positions enhance overall enzyme stability and proteolytic resistance without compromising the catalytic function, thereby resolving the contradiction between reliability and productivity
2Reliability
If PAL is used as enzyme therapy, then phenylalanine metabolism can be improved, but immunogenicity and proteolytic sensitivity increase
Solution Approach 1:
The patent applies parameter changes by modifying the immunogenic epitopes of PAL through amino acid substitutions at positions C64, C318, C503, and C565. These changes alter the enzyme's immunogenic properties while preserving its catalytic function, thereby reducing immunogenicity and proteolytic sensitivity while maintaining therapeutic efficacy
Solution Approach 2:
The patent creates variant copies of the wild-type PAL enzyme with modified amino acid sequences. These variant copies retain the essential catalytic function for phenylalanine metabolism but have reduced immunogenicity due to amino acid substitutions, effectively copying the therapeutic function while eliminating harmful immunogenic properties
3Productivity
If PAL variants with amino acid substitutions are developed, then catalytic activity and thermal stability improve, but protein aggregation may increase
Solution Approach 1:
The patent applies parameter changes by systematically testing combinations of amino acid substitutions (C503S/C565L, C503S/C565P, C503L/C565P, and others) to optimize the balance between catalytic activity and aggregation resistance. The selected variants achieve enhanced kcat/Km ratios and thermal stability while maintaining low aggregation propensity through careful selection of amino acid changes at critical positions
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The PAL variants exhibit increased catalytic activity, improved thermal stability, and lower aggregation, making them more effective for treating conditions like PKU.
Implementation Method 1
Phenylalanine ammonia lyase (PAL) is a member of the aromatic amino acid lyase family (EC 4.3.1.23-1.25 and 4.3.1.3) together with histidine ammonia lyase (HAL) and tyrosine ammonia lyase (TAL). More specifically, enzymes with PAL activity (EC 4.3.1.23-1.25 and formerly classified as EC 4.3.1.5) catalyze the nonoxidative deamination of L-phenylalanine to trans-cinnamic acid.
Implementation Method 2
The PAL variants according to the present disclosure exhibit increased catalytic activity, improved thermal stability, and lower aggregation, making them more effective for treating conditions like PKU.
Data Source
AI summary
The present disclosure provides a variant of phenylalanine ammonia lyase (PAL), the phenylalanine ammonia lyase (PAL) being derived from Anabaena variabilis and comprises the amino acid sequence shown in SEQ ID NO: 1, wherein the variant comprises amino acid substitution C503S/C565L, so that the variant comprises the amino acid sequence shown in SEQ ID NO: 2; or amino acid substitution C503S/C565P, so that the variant comprises the amino acid sequence shown in SEQ ID NO: 3; or amino acid substitution C503L/C565P, so that the variant comprises the amino acid sequence shown in SEQ ID NO: 4. The present disclosure also provides a conjugate and a pharmaceutical composition comprising the variant, a polynucleotide encoding the variant, a bacterium comprising the variant, and a method for preparing the variant.