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The Thioesterase Bhp is Involved in the Formation of β‐Hydroxytyrosine during Balhimycin Biosynthesis in Amycolatopsis balhimycina
Author(s) -
Mulyani Sri,
Egel Ellen,
Kittel Claudia,
Turkanovic Suada,
Wohlleben Wolfgang,
Süssmuth Roderich D.,
van Pée KarlHeinz
Publication year - 2010
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200900600
Subject(s) - thioesterase , chemistry , thioester , esterase , biochemistry , biosynthesis , hydrolysis , hydrolase , enzyme , escherichia coli , stereochemistry , gene
The putative hydrolase gene bhp from the balhimycin biosynthetic gene cluster has been cloned and overexpressed in Escherichia coli . The corresponding enzyme Bhp was purified to homogeneity by nickel‐chelating chromatography and characterized. Although Bhp has sequence similarities to hydrolases with “haloperoxidase”/perhydrolase activity, it did not show any enzymatic activity with standard “haloperoxidase”/perhydrolase substrates (e.g., monochlorodimedone and phenol red), nonspecific esterase substrates (such as p ‐nitrophenyl acetate, p ‐nitrophenyl phosphate and S ‐thiophenyl acetate) or the model lactonase substrate dihydrocoumarin. However, Bhp could be shown to catalyse the hydrolysis of S ‐β‐hydroxytyrosyl‐ N ‐acetyl cysteamine thioester (β‐OH‐Tyr‐SNAC) with 15 times the efficiency of S ‐ L ‐tyrosyl‐ N ‐acetyl cysteamine thioester ( L ‐Tyr‐SNAC). This is in agreement with the suggestion that Bhp is involved in balhimycin biosynthesis, during which it was supposed to catalyse the hydrolysis of β‐OH‐Tyr‐ S ‐PCP (PCP=peptidyl carrier protein) to free β‐hydroxytyrosine (β‐OH‐Tyr) and strongly suggests that Bhp is a thioesterase with high substrate specificity for PCP‐bound β‐OH‐Tyr and not a “haloperoxidase”/perhydrolase or nonspecific esterase.