Literature summary for 4.2.3.46 extracted from

Green, S.; Squire, C.J.; Nieuwenhuizen, N.J.; Baker, E.N.; Laing, W.
Defining the potassium binding region in an apple terpene synthase (2009), J. Biol. Chem., 284, 8661-8669.

Cloned(Commentary)

Cloned (Comment)	Organism
mutated enzymes are generated using the QuickChange II site-directed mutagenesis kit. The PCR-based mutagenesis protocol is performed using pET-30a harbouring the MdAFS1 cDNAs as template. The single-site mutant enzymes overexpressed in Escherichia coli.	Malus domestica

Protein Variants

Protein Variants	Comment	Organism
D484A	85% loss of sesquiterpene synthase activity compared with wild-type enzyme when K+ is present	Malus domestica
D484A	85% loss of sesquiterpene synthase activity compared with wild-type enzyme. Little change in K+-independent activity	Malus domestica
S485A	exhibits marginally increased sesquiterpene synthase activities, and an approximate 2fold increase in monoterpene synthase activity compared with the wild-type enzyme	Malus domestica
S485A	mutant exhibits marginally increased sesquiterpene synthase activities, and an approximate 2fold increase in monoterpene synthase activity compared with the wild-type enzyme	Malus domestica
S487A	95% decrease in sesquiterpene synthase activity compared with wild-type enzyme when K+ is present	Malus domestica
S487A	95% decrease in sesquiterpene synthase activity compared with wild-type enzyme. Little change in K+-independent activity	Malus domestica
S487K	The S487K mutant has 35-45% of the wild-type activity with farnesyl diphosphate, with no significant alterations in the alpha-farnesene isomer ratios produced and a small decrease in catalytic efficiency	Malus domestica
S487K	mutant has 3545% of the wild-type activity with farnesyl diphosphate, with no significant alterations in the alpha-farnesene isomer ratios produced and a small decrease in catalytic efficiency	Malus domestica
S488A	mutant shows decreases in both sesqui- and monoterpene synthase activities compared with the WT enzyme, with mono-TPS activity being reduced more than sesquiterpene synthase activity. Sesquiterpene synthase (alpha-farnesene) products produced by the mutated and wild-type enzymes are identical, there are no significant alterations in the ratios of the alpha-farnesene isomers produced.	Malus domestica
S488A	mutant shows decreases in both sesqui- and mono-terpene synthases activities, compared with the wild-type enzyme, with mono-terpene synthases activity being reduced more than sesqui-terpene synthases activity	Malus domestica

Metals/Ions

Metals/Ions	Comment	Organism
K+	MdAFS1 retains up to 12% of its activity in the absence of K+, enzyme contains K+ binding region, MdAFS1 exhibits a type II K+ response, MdAFS1 is not absolutely dependent upon M+ its unequivocal classification as type I or type II K+ activated, or that of any other terpene synthases, will not be possibl, then type I enzymes can exhibit type II kinetics and vice versa.	Malus domestica
K+	activity is dependent on K+, the potassium binding region is defined	Malus domestica
Mg2+	10 mM are included in farnesyl diphosphate activity assay	Malus domestica

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
(2E,6E)-farnesyl diphosphate	Malus domestica	commentary	(3E,6E)-alpha-farnesene + diphosphate	-	?
additional information	Malus domestica	the monoterpene synthase ((E)-beta-ocimene and beta-myrcene) and sesquiterpene synthase (alpha-farnesene) products produced by the mutated and wild-type enzymes are identical, there are no significant alterations in the ratios of the alpha-farnesene isomers produced	?	-	?

Organism

Organism	UniProt	Comment	Textmining
Malus domestica	B2ZZ11	-	-
Malus domestica	Q84LB2	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant MdAFS1protein is extracted and purified	Malus domestica

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
additional information	-	S487K mutant has 35-45% of the wild-type activity with farnesyl diphosphate with no significant alterations in the alpha-farnesene isomer ratios produced and a small decrease in catalytic efficiency (wild-type and S487K respective kcat/Km values of 17.5 and 13.3 mM/s)	Malus domestica

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
(2E,6E)-farnesyl diphosphate	-	Malus domestica	(3E,6E)-alpha-farnesene + diphosphate	-	?
(2E,6E)-farnesyl diphosphate	commentary	Malus domestica	(3E,6E)-alpha-farnesene + diphosphate	-	?
geranyl diphosphate	poor substrate	Malus domestica	(E)-beta-ocimene + beta-myrcene	-	?
additional information	the monoterpene synthase ((E)-beta-ocimene and beta-myrcene) and sesquiterpene synthase (alpha-farnesene) products produced by the mutated and wild-type enzymes are identical, there are no significant alterations in the ratios of the alpha-farnesene isomers produced	Malus domestica	?	-	?

Synonyms

Synonyms	Comment	Organism
alpha-farnesene synthase	-	Malus domestica
MdAFS1	-	Malus domestica

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	farnesyl diphosphate and geranyl diphosphate activity assay	Malus domestica

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.000005	-	geranyl diphosphate	mutant S487A	Malus domestica
0.000005	-	geranyl diphosphate	pH 7.5, 30°C, mutant enzyme S487A	Malus domestica
0.000034	-	geranyl diphosphate	mutant D484A	Malus domestica
0.000034	-	geranyl diphosphate	pH 7.5, 30°C, mutant enzyme D484A	Malus domestica
0.000052	-	geranyl diphosphate	mutant S488A	Malus domestica
0.000052	-	geranyl diphosphate	pH 7.5, 30°C, mutant enzyme S488A	Malus domestica
0.000281	-	geranyl diphosphate	wild-type	Malus domestica
0.000281	-	geranyl diphosphate	pH 7.5, 30°C, wild-type enzyme	Malus domestica
0.000588	-	geranyl diphosphate	mutant S485A	Malus domestica
0.000588	-	geranyl diphosphate	pH 7.5, 30°C, mutant enzyme S485A	Malus domestica
0.0026	-	farnesyl diphosphate	mutant S487A, 50 mM KCl, 10 mM MgCl2	Malus domestica
0.0026	-	(2E,6E)-farnesyl diphosphate	pH 7.5, 30°C, mutant enzyme S487A	Malus domestica
0.0062	-	farnesyl diphosphate	without K+	Malus domestica
0.0086	-	farnesyl diphosphate	mutant D484A, 50 mM KCl, 10 mM MgCl2	Malus domestica
0.0086	-	(2E,6E)-farnesyl diphosphate	pH 7.5, 30°C, mutant enzyme D484A	Malus domestica
0.022	-	farnesyl diphosphate	without K+, sesquiterpene synthase activity in mutant S487K is independent of K+	Malus domestica
0.039	-	farnesyl diphosphate	mutant S488A, 50 mM KCl, 10 mM MgCl2	Malus domestica
0.039	-	(2E,6E)-farnesyl diphosphate	pH 7.5, 30°C, mutant enzyme S488A	Malus domestica
0.0533	-	farnesyl diphosphate	wild-type MdAFS, 50 mM KCl, 10 mM MgCl2	Malus domestica
0.0553	-	(2E,6E)-farnesyl diphosphate	pH 7.5, 30°C, wild-type enzyme	Malus domestica
0.0613	-	farnesyl diphosphate	mutant S485A, 50 mM KCl, 10 mM MgCl2	Malus domestica
0.0613	-	(2E,6E)-farnesyl diphosphate	pH 7.5, 30°C, mutant enzyme S485A	Malus domestica

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	farnesyl diphosphate activity assay	Malus domestica