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Removing a bottleneck in the Bacillus subtilis biotin pathway: BioA utilizes lysine rather than S ‐adenosylmethionine as the amino donor in the KAPA‐to‐DAPA reaction
Author(s) -
Van Arsdell Scott W.,
Perkins John B.,
Yocum R. Rogers,
Luan Linda,
Howitt C. Linda,
Prasad Chatterjee Nilu,
Pero Janice G.
Publication year - 2005
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.20488
Subject(s) - lysine , bacillus subtilis , amino acid , escherichia coli , biotin , biochemistry , biosynthesis , biology , serine , fermentation , enzyme , chemistry , gene , bacteria , genetics
In biotin biosynthesis, DAPA aminotransferase encoded by the bioA gene catalyzes the formation of the intermediate 7,8‐diaminopelargonic acid (DAPA) from 7‐keto‐8‐aminopelargonic acid (KAPA). DAPA aminotransferases from Escherichia coli , Serratia marcescens , and Bacillus sphaericus use S ‐adenosylmethionine (SAM) as the amino donor. Our observation that SAM is not an amino donor for B. subtilis DAPA aminotransferase led to a search for an alternative amino donor for this enzyme. Testing of 26 possible amino acids in a cell‐free extract assay revealed that only l ‐lysine was able to dramatically stimulate the in vitro conversion of KAPA to DAPA by the B. subtilis DAPA aminotransferase. The K m for lysine and KAPA was estimated to be between 2 and 25 mM, which is significantly higher than the K m of purified E. coli BioA for SAM (0.15 mM). This higher requirement for lysine resulted in accumulation of KAPA during fermentation of B. subtilis biotin producing strains. However, this pathway bottleneck could be relieved by either addition of exogenous lysine to the medium or by introduction of lysine deregulated mutations into the production strains. © 2005 Wiley Periodicals, Inc.