Information on EC 1.14.14.95 - germacrene A hydroxylase

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EC 1.1.1.314

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formerly

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EC 1.14.13.123

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formerly

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GAO

germacrene A oxidase

NADP+-dependent sesquiterpenoid dehydrogenase

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(+)-germacrene A + 3 [reduced NADPH-hemoprotein reductase] + 3 O2 = germacra-1(10),4,11(13)-trien-12-oate + 3 [oxidized NADPH-hemoprotein reductase] + 4 H2O

overall reaction

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(+)-germacrene A + O2 + [reduced NADPH-hemoprotein reductase] = germacra-1(10),4,11(13)-trien-12-ol + [oxidized NADPH-hemoprotein reductase] + H2O

(1a)

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germacra-1(10),4,11(13)-trien-12-al + O2 + [reduced NADPH-hemoprotein reductase] = germacra-1(10),4,11(13)-trien-12-oate + [oxidized NADPH-hemoprotein reductase] + H2O

(1c)

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germacra-1(10),4,11(13)-trien-12-ol + O2 + [reduced NADPH-hemoprotein reductase] = germacra-1(10),4,11(13)-trien-12-al + [oxidized NADPH-hemoprotein reductase] + 2 H2O

(1b)

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hydroxylation

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C13-hydroxylation

sesquiterpene lactone biosynthesis

BRENDA

KEGG

Biosynthesis of secondary metabolites, Sesquiterpenoid and triterpenoid biosynthesis

(+)-germacrene-A,[reduced NADPH-hemoprotein reductase]:oxygen oxidoreductase (12-hydroxylating)

A cytochrome P-450 (heme-thiolate) protein. This plant enzyme catalyses three steps in a pathway that leads to the biosynthesis of many sesquiterpenoid lactones.

(+)-germacrene A + [reduced NADPH-hemoprotein reductase] + O2

germacra-1(10),4,11(13)-trien-12-ol + [oxidized NADPH-hemoprotein reductase] + H2O

2 entries

beta-elemene + [reduced NADPH-hemoprotein reductase] + O2

elema-1,3,11(13)-trien-12-ol + [oxidized NADPH-hemoprotein reductase] + H2O

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Substrates: the amount of (+)-beta-elemene hydroxylated is 2times less than that of (-)-beta-elemene
Products: -

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germacra-1(10),4,11(13)-trien-12-ol + 2 O2 + 2 [reduced NADPH-hemoprotein reductase]

germacra-1(10),4,11(13)-trien-12-oate + 2 [oxidized NADPH-hemoprotein reductase] + 3 H2O

2 entries

germacrene A + [reduced NADPH-hemoprotein reductase] + O2

12-hydroxygermacrene A + [oxidized NADPH-hemoprotein reductase] + H2O

cytochrome P-450

6 entries

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flavin

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an assay buffer without flavins (FAD and FMN) gives 18% loss of hydroxylase activity. However, omitting these flavins from the extraction buffer results in a loss of more than 70% in enzyme activity

NADP+

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dependent on

aminobenzotriazole

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26% inhibition at 0.1 mM

carbon monoxide

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blue-light reversible inhibition, an atmosphere of 80% CO plus 20% O2 inhibits (+)-germacrene A hydroxylase by 69%

clotrimazole

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016% inhibition at 0.1 mM

cytochrome c

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97% inhibition at 0.1 mM

Metyrapone

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23% inhibition at 1 mM

miconazole

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30% inhibition at 0.1 mM

additional information

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flushing the reaction mixture for 1.5 min with argon prior to incubation causes a 69% decrease of enzyme activity because of O2 depletion

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8

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6 - 9

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60% of maximal enzyme activity at pH 7.5 and 9.0 (no difference in activity between bis-Tris and Tris buffer)

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UniProt

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UniProt

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UniProt

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UniProt

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Highest Expressing Human Cell Lines

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Cell Line Links

Gene Links

54900

expressed in Saccharomyces cerevisiae strain YPL 154C:Pep4 KO

Lactuca sativa

D5J9U8

715506

expressed in the protease-deficient Saccharomyces cerevisiae YPL 154C:Pep4 KO strain