HPPD Enzyme Mutations for Herbicide Tolerance
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Solution Overview
Problem
Current HPPD inhibitor herbicides exhibit slow-binding or slow, tight-binding properties, making it challenging to develop plants fully tolerant to these herbicides, as mutations that reduce inhibitor affinity do not completely overcome inhibition due to the persistent binding.
Innovation Solution
Designing HPPD enzymes with reduced affinity and increased dissociation rate of HPPD inhibitors, converting them from slow-binding inhibitors to fully reversible inhibitors, thereby achieving maximized tolerance.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If mutations are introduced to reduce inhibitor affinity, then herbicide tolerance is improved, but slow-binding inhibition persists due to persistent binding
Solution Approach 1:
The patent applies parameter changes by modifying the HPPD enzyme's kinetic parameters through site-directed mutagenesis. Specifically, mutations at positions 335 and 336 change the enzyme's affinity (Km) and catalytic efficiency (kcat) for HPPD inhibitors, transforming the inhibition mechanism from slow-binding to reversible while maintaining herbicide tolerance
Solution Approach 2:
The patent introduces dynamics by changing the time-dependent binding characteristics of the HPPD-inhibitor complex. The mutations convert a static slow-binding interaction into a dynamic reversible interaction, allowing the inhibitor to bind and dissociate rapidly rather than persisting on the enzyme
2Reliability
If HPPD enzyme affinity for inhibitors is reduced, then tolerance to HPPD-inhibitors is improved, but enzyme catalytic efficiency may be affected
Solution Approach 1:
The patent applies local quality by making specific localized changes at positions 335 and 336 of the HPPD enzyme while leaving the rest of the catalytic domain unchanged. This localized mutagenesis approach selectively modifies inhibitor binding properties without disrupting the overall catalytic function of the enzyme
Solution Approach 2:
The patent changes kinetic parameters (Km and kcat) through targeted mutations, optimizing the balance between inhibitor tolerance and catalytic efficiency. The mutations adjust these parameters to achieve full tolerance while preserving sufficient enzymatic activity for plant metabolism
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.