4.2.3.119: (-)-alpha-pinene synthase
This is an abbreviated version!
For detailed information about (-)-alpha-pinene synthase, go to the full flat file.
Word Map on EC 4.2.3.119
Reaction
Synonyms
(-)-alpha-pinene cyclase, (-)-alpha-pinene/(-)-camphene synthase, (-)-limonene/(-)-alpha-pinene synthase, (-)-pinene cyclase, (-)-pinene synthase, ag6, ag9, alpha pinene sythase, alpha-pinene sythase, AvPS, AvTPS1, camphene synthase, cyclase II, EC 4.1.99.8, EC 4.2.3.14, LPS, mono-tps, More, pinene synthase, pinene-synthase, PlPIN, PT1, QH6, TPS-(-)apin1, TPS-(-)bpin2, TPS1, TPS2, TPS3
ECTree
4.2.3.119 (-)-alpha-pinene synthaseAdvanced search results
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Engineering on EC 4.2.3.119 - (-)-alpha-pinene synthase
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PROTEIN VARIANTS 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
C372S
replacement with corresponding residue of (-)-camphene synthase, 97% of wild-type activity. Mutant produces an increased proportion of (-)-alpha-pinene and a correspondingly decreased proportion of (-)-beta-pinene, while the levels of total pinenes remains relatively constant
C372S/C480S
replacement with corresponding residue of (-)-camphene synthase, 72% of wild-type activity. Mutant produces an increased proportion of (-)-alpha-pinene and a correspondingly decreased proportion of (-)-beta-pinene
C372S/F597W
replacement with corresponding residue of (-)-camphene synthase, 100% of wild-type activity. Mutant produces an increased proportion of (-)-alpha-pinene and a correspondingly decreased proportion of (-)-beta-pinene
C372S/F597W/S485C/F597W
replacement with corresponding residue of (-)-camphene synthase, 99% of wild-type activity. Mutant produces about 80%(-)-alpha-pinene and 10% (-)-beta-pinene
C372S/S485C
replacement with corresponding residue of (-)-camphene synthase, 92% of wild-type activity. Mutant produces an increased proportion of (-)-alpha-pinene and a correspondingly decreased proportion of (-)-beta-pinene
C480S
replacement with corresponding residue of (-)-camphene synthase, 97% of wild-type activity. Mutant produces an increased proportion of (-)-alpha-pinene and a correspondingly decreased proportion of (-)-beta-pinene, while the levels of total pinenes remains relatively constant
C480S/F597W
replacement with corresponding residue of (-)-camphene synthase, 7% of wild-type activity. Mutant produces an increased proportion of (-)-alpha-pinene and a correspondingly decreased proportion of (-)-beta-pinene
C480S/S485C
replacement with corresponding residue of (-)-camphene synthase, 70% of wild-type activity. Mutant produces an increased proportion of (-)-alpha-pinene and a correspondingly decreased proportion of (-)-beta-pinene
F597W
replacement with corresponding residue of (-)-camphene synthase, 73% of wild-type activity. Mutant produces an increased proportion of (-)-alpha-pinene and a correspondingly decreased proportion of (-)-beta-pinene, while the levels of total pinenes remains relatively constant
S485C
replacement with corresponding residue of (-)-camphene synthase, 100% of wild-type activity. Mutant produces an increased proportion of (-)-alpha-pinene and a correspondingly decreased proportion of (-)-beta-pinene, while the levels of total pinenes remains relatively constant
S485C/F597W
replacement with corresponding residue of (-)-camphene synthase, 68% of wild-type activity. Mutant produces an increased proportion of (-)-alpha-pinene and a correspondingly decreased proportion of (-)-beta-pinene
H346Y
site-directed mutagenesis, mutation in the alpha-domain (catalytic domain), no phenotype, similar to wild-type
Q456K
site-directed mutagenesis, mutation in the alpha-domain (catalytic domain), the mutant shows increased catalytic activity compared to the wild-type
Q456L
Q456P
site-directed mutagenesis, mutation in the alpha-domain (catalytic domain), the mutant shows 50% reduced catalytic activity compared to the wild-type
Q456V
site-directed mutagenesis, mutation in the alpha-domain (catalytic domain), the mutant shows increased catalytic activity compared to the wild-type
additional information
Q456L
site-directed mutagenesis, mutation in the alpha-domain (catalytic domain), the mutant shows a reduction in pigmentation (PSmut) but shows improved catalytic activity
Q456L
site-directed mutagenesis, mutation in the alpha-domain (catalytic domain), the mutant shows increased catalytic activity compared to the wild-type
additional information
replacement of selected amino acid residues in (-)-pinene synthase with the corresponding residues from (-)-camphene synthase in an effort to identify the amino acids responsible for the catalytic diVerences. The approach produces an enzyme in which more than half of the product is channeled through an alternative pathway. Several (-)-pinene synthase to (-)-camphene synthase amino acid substitutions are necessary before catalysis is significantly altered
additional information
to elucidate critical amino acids involved in determining monoterpene product distribution, a combination of domain swapping and reciprocal site-directed mutagenesis was carried out between (-)-(4S)-limonene synthase LS and (-)-(4S)-limonene/(-)-(1S, 5S)-alpha-pinene synthase LPS. Amino acids in the predicted D through F helix regions are critical for product determination. Chimera consisting of N-terminal 218 residues of LS plus corresponding C-terminus of LPS produces 20.7% alpha-pinene, 11.2% sabinene, 7.1% beta-pinene, 25.6% limonene, 35.4% beta-phellandrene, with 42.8% relative activity. Chimera consisting of N-terminal 518 residues of LS plus corresponding C-terminus of LPS produces 6.4% alpha-pinene, 1.5% sabinene, 11% beta-pinene, 64.1% limonene, 17% beta-phellandrene, with 101% relative activity. Chimera consisting of N-terminal 442 residues of LPS plus corresponding C-terminus of LS produces 11% alpha-pinene, 1.4% sabinene, 6.3% beta-pinene, 47.9% limonene, 33.3% beta-phellandrene, with 41.7% relative activity
additional information
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to elucidate critical amino acids involved in determining monoterpene product distribution, a combination of domain swapping and reciprocal site-directed mutagenesis was carried out between (-)-(4S)-limonene synthase LS and (-)-(4S)-limonene/(-)-(1S, 5S)-alpha-pinene synthase LPS. Amino acids in the predicted D through F helix regions are critical for product determination. Chimera consisting of N-terminal 218 residues of LS plus corresponding C-terminus of LPS produces 20.7% alpha-pinene, 11.2% sabinene, 7.1% beta-pinene, 25.6% limonene, 35.4% beta-phellandrene, with 42.8% relative activity. Chimera consisting of N-terminal 518 residues of LS plus corresponding C-terminus of LPS produces 6.4% alpha-pinene, 1.5% sabinene, 11% beta-pinene, 64.1% limonene, 17% beta-phellandrene, with 101% relative activity. Chimera consisting of N-terminal 442 residues of LPS plus corresponding C-terminus of LS produces 11% alpha-pinene, 1.4% sabinene, 6.3% beta-pinene, 47.9% limonene, 33.3% beta-phellandrene, with 41.7% relative activity
additional information
estalishment of a pinene production system in recombinant Escherichia coli by coexpression of (-)-alpha-pinene synthase from Pinus paeda and Abies grandis GPPS, as well as farnesyl diphosphate synthase mutant IspA(S80F) from Escherichia coli. The isolated alpha-pinene synthase variant PSmut outperforms the wild-type (parent) enzyme in multiple contexts in Escherichia coli and cyanobacteria. The purified variant exhibits drastically altered metal dependency, enabling to keep the activity in the cytosol that is manganese-deficient. Coexpression of this variant with mevalonate pathway enzymes, isopentenyl diphosphate isomerase, and GPP synthase yield 140 mg/l pinene in a flask culture. Screening for PS mutants with higher cellular activity and production method optimization, overview
