4.2.3.119	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	663770	
4.2.3.119	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	663770	
4.2.3.119	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	663770	
4.2.3.119	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	663770	
4.2.3.119	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	663770	
4.2.3.119	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	663770	
4.2.3.119	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	663770	
4.2.3.119	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	663770	
4.2.3.119	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	663770	
4.2.3.119	H346Y	site-directed mutagenesis, mutation in the alpha-domain (catalytic domain), no phenotype, similar to wild-type	746601	
4.2.3.119	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	746601	
4.2.3.119	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	663770	
4.2.3.119	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	649494	
4.2.3.119	Q456K	site-directed mutagenesis, mutation in the alpha-domain (catalytic domain), the mutant shows increased catalytic activity compared to the wild-type	747323	
4.2.3.119	Q456L	site-directed mutagenesis, mutation in the alpha-domain (catalytic domain), the mutant shows a reduction in pigmentation (PSmut) but shows improved catalytic activity	746601	
4.2.3.119	Q456L	site-directed mutagenesis, mutation in the alpha-domain (catalytic domain), the mutant shows increased catalytic activity compared to the wild-type	747323	
4.2.3.119	Q456P	site-directed mutagenesis, mutation in the alpha-domain (catalytic domain), the mutant shows 50% reduced catalytic activity compared to the wild-type	747323	
4.2.3.119	Q456V	site-directed mutagenesis, mutation in the alpha-domain (catalytic domain), the mutant shows increased catalytic activity compared to the wild-type	747323	
4.2.3.119	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	663770	
4.2.3.119	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	663770