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Characterization of the Aminotransferase ThdN from Thienodolin Biosynthesis in Streptomyces albogriseolus
Author(s) -
Milbredt Daniela,
Patallo Eugenio P.,
van Pée KarlHeinz
Publication year - 2016
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.201600304
Subject(s) - tryptophan , biosynthesis , stereochemistry , enzyme , indole test , pseudomonas fluorescens , biochemistry , streptomyces , pyridoxal , chemistry , enzyme kinetics , pseudomonas , biology , amino acid , bacteria , active site , genetics
In Streptomyces albogriseolus the indolethiophen alkaloid thienodolin is derived from tryptophan. The first step in thienodolin biosynthesis is the regioselective chlorination of tryptophan in the 6‐position of the indole ring. The second step is catalyzed by the aminotransferase ThdN. ThdN shows sequence homology (up to 69 % similarity) with known pyridoxal 5′‐phosphate‐dependent aminotransferases of the aspartate aminotransferase family from Gram‐positive bacteria. thdN was heterologously expressed in Pseudomonas fluorescens , and the enzyme was purified by nickel‐affinity chromatography. ThdN is a homodimeric enzyme with a mass of 90 600 kDa and catalyzes the conversion of l ‐tryptophan and a number of chlorinated and brominated l ‐tryptophans. The lowest K M values were found for 6‐bromo‐ and 6‐chlorotryptophan (40 and 66 μ m , respectively). For l ‐tryptophan it was 454 μ m, which explains why thienodolin is the major product and dechlorothienodolin is only a minor component. The turnover number ( k cat ) for 7‐chlorotryptophan (128 min −1 ) was higher than that for the natural substrate 6‐chlorotryptophan (88 min −1 ).