HPPD Enzyme Mutations for Herbicide Tolerance
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Solution Overview
Problem
Current HPPD inhibitor herbicides act as slow-binding or slow, tight-binding inhibitors, making it difficult to design HPPD enzymes tolerant to these inhibitors, as mutations that reduce affinity do not fully overcome inhibition due to the inhibitors' kinetic properties, resulting in incomplete tolerance.
Innovation Solution
Designing HPPD enzymes with increased dissociation rates of slow-binding inhibitors, reducing their affinity and making them fully reversible, thereby overcoming the challenges of slow dissociation and long-lasting inhibition.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If mutations are introduced to reduce HPPD enzyme affinity for HPPD inhibitors, then herbicide tolerance is improved, but the inhibitors' slow-binding kinetic properties cause incomplete tolerance due to prolonged inhibition
Solution Approach 1:
The patent applies parameter changes by modifying the kinetic parameters of the HPPD enzyme-inhibitor interaction. Specifically, it introduces mutations that alter the dissociation rate constant (koff) to increase the dissociation rate of slow-binding inhibitors from the enzyme active site. This changes the inhibition from slow-reversing to fast-reversing, allowing the enzyme to regain activity more quickly after inhibitor binding, thereby achieving complete herbicide tolerance despite the inhibitors' inherent slow-binding properties
2Adaptability or versatility
If HPPD enzyme affinity for HPPD inhibitors is reduced through mutation, then enzyme tolerance increases, but slow dissociation rates result in long-lasting inhibition
Solution Approach 1:
The patent applies dynamics by making the enzyme-inhibitor interaction reversible and time-dependent in a controlled manner. Through mutations at specific residues (e.g., Asn-336, Gln-339, Thr-340 in Arabidopsis HPPD), the enzyme is engineered to rapidly release inhibitors after binding, creating a dynamic system where inhibition is temporary rather than persistent. This allows the enzyme to adapt quickly to the presence of inhibitors by entering and exiting the inhibited state rapidly, achieving versatility in tolerating HPPD inhibitor herbicides
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
Achieves full tolerance to HPPD inhibitors by reducing the enzymes' affinity and increasing the dissociation rate, ensuring the inhibitors are no longer slow-binding, allowing for effective herbicide application without long-term enzyme inhibition.
Implementation Method 1
The 4-hydroxyphenylpyruvate dioxygenases (HPPDs) are enzymes which catalyze the reaction in which para-hydroxyphenylpyruvate (abbreviated herein as HPP), a tyrosine degradation product, is transformed into homogentisate (abbreviated herein as HG)
Data Source
AI summary
In the present invention, HPPD enzymes and plants containing them showing a full tolerance against several classes of HPPD-inhibitors are described. A set of HPPD enzymes have been designed which have either no or only a significantly reduced affinity to HPPD inhibitors and, at the same time, the rate of dissociation of the HPPD inhibitors of the enzyme is increased to such an extent that the HPPD inhibitors no longer act as slow-binding or slow, tight-binding inhibitors but, instead of this, have become fully reversible inhibitors. In particular, isolated polynucleotides encoding HPPD inhibitor tolerance polypeptides are provided. Additionally, amino acid sequences corresponding to the polynucleotides are encompassed.