Mutant OPAA enzymes with increased catalytic efficiency on organophosphorus compound EA1356
Inventors
Harvey, Steven P • Guelta, Mark A • McMahon, Leslie R
Assignees
United States Department of the Army • Government of the United States of America
Publication Number
US-10124043-B1
Publication Date
2018-11-13
Expiration Date
2038-02-14
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Abstract
The invention comprises isolated, mutant, non-wild-type organophosphorus acid anhydrolase (OPAA) enzymes having three site mutations, methods of production, and methods of use to effectively degrade organophosphorus compound EA1356 (2-methylcyclohexyl methylphosphonofluoridate) with greater catalytic efficiency than the wild-type OPAA enzyme.
Core Innovation
The invention relates to isolated, mutant, non-wild-type organophosphorus acid anhydrolase (OPAA) enzymes comprising mutations at sequence positions 212, 342, and 215 of SEQ ID NO: 1. These mutants have amino acid substitutions that enhance catalytic efficiency specifically for degrading the toxic organophosphorus compound EA1356 (2-methylcyclohexyl methylphosphonofluoridate). A preferred mutant, designated FLH, includes the substitutions Y212F, V342L, and I215H, and exhibits approximately four times greater catalytic efficiency than the wild-type enzyme.
The background identifies a significant problem: organophosphorus compounds like EA1356 are highly toxic and represent hazards to human health and the environment. While native OPAA enzymes have some catalytic activity against various organophosphorus nerve agents, their catalytic efficiency against EA1356 is limited, rendering them marginally useful for decontamination or treatment of poisoning by this compound. There was no efficient, easily produced catalyst known for EA1356 degradation prior to this invention.
The invention solves this problem by providing mutant OPAA enzymes with specific amino acid substitutions at positions 212, 342, and 215 that significantly enhance the catalytic efficiency on EA1356. The document discloses methods of producing and purifying these mutant enzymes, pharmaceutical compositions containing them, and methods of use including catalytic degradation of EA1356. The mutants retain the functional activity of the wild-type enzyme but offer improved degradation performance on this toxic substrate.
Claims Coverage
The patent includes two independent claims directed to the mutant OPAA enzyme and methods for degrading EA1356 using this mutant enzyme. These define the core inventive features involving specific mutations and their application for detoxification.
Mutant OPAA enzyme with specific triplet mutations
An isolated OPAA enzyme comprising non-wild-type amino acids at each of positions 212, 342, and 215 of SEQ ID NO: 1.
Specific amino acid substitutions at key positions
The non-wild-type amino acid at position 212 is selected from Phenylalanine (F), Leucine (L), Isoleucine (I), Glutamine (Q), and Tyrosine (Y). The non-wild-type amino acid at position 342 is selected from Leucine (L), Isoleucine (I), Glutamine (Q), Proline (P), and Tyrosine (Y). The non-wild-type amino acid at position 215 is selected from Histidine (H), Leucine (L), Threonine (T), Cysteine (C), Arginine (R), and Lysine (K).
Preferred mutant sequence FLH
A mutant OPAA comprising the amino acid sequence of SEQ ID NO: 2, specifically Y212F, V342L, and I215H mutations.
Method of degrading EA1356 using mutant OPAA
Contacting EA1356 with a mutant OPAA having the specified positions mutated to non-wild-type amino acids, including the preferred FLH mutant sequence.
Therapeutic use of mutant OPAA for EA1356 poisoning
Administering a pharmaceutical composition containing the mutant OPAA, e.g., the FLH mutant, to a subject to degrade EA1356 in vivo.
Kit for degrading EA1356
A kit comprising the isolated mutant OPAA enzyme with mutations at positions 212, 342, and 215 of SEQ ID NO: 1, optionally with pharmaceutically acceptable carriers and adjuvants.
The claims collectively cover mutant OPAA enzymes with targeted amino acid substitutions at positions 212, 342, and 215 that enhance catalytic efficiency on EA1356, methods to degrade EA1356 using these mutants including therapeutic administration, and kits containing the mutant enzymes for such applications.
Stated Advantages
The mutant OPAA enzyme exhibits approximately a four-fold increase in catalytic efficiency on the toxic organophosphorus compound EA1356 compared to the wild-type enzyme.
The enhanced catalytic activity allows for effective catalytic degradation of EA1356, useful for in vivo treatment of poisoning and environmental or surface decontamination.
Mutations narrow the size of the enzyme's substrate-binding small pocket, favoring an orientation of EA1356 that is more conducive to catalysis of its P—F bond.
The mutant enzymes can be efficiently expressed, purified, formulated, and administered using a variety of delivery methods and pharmaceutical compositions.
Documented Applications
In vivo treatment of EA1356 poisoning in subjects by administering pharmaceutical compositions containing the mutant OPAA enzyme.
Catalytic decontamination of EA1356 from contaminated surfaces or environmental sources by applying the mutant OPAA enzyme.
Pharmaceutical use comprising delivery of mutant OPAA via various routes including intravenous, subcutaneous, intramuscular, inhalation, and others for therapeutic effect.
Incorporation of the mutant OPAA enzyme into kits with pharmaceutically acceptable carriers and optional boosters or applicators for degradation of EA1356.
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