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.
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.
Van Arsdell
Scott W
SW
OmniGene Bioproducts, Inc., 763D Concord Ave., Cambridge, Massachusetts 02138, USA.
Perkins
John B
JB
Yocum
R Rogers
RR
Luan
Linda
L
Howitt
C Linda
CL
Chatterjee
Nilu Prasad
NP
Pero
Janice G
JG
D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D000599:Amino Acids, Diamino / Q000235:genetics / Q000378:metabolism*; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001710:Biotin / Q000096:biosynthesis*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D008239:Lysine / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis; D012436:S-Adenosylmethionine / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D000637:Transaminases / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D000599:Amino Acids, Diamino; D001426:Bacterial Proteins; D011994:Recombinant Proteins; C004720:7,8-diaminopelargonic acid; C001502:7-keto-8-aminopelargonic acid; D001710:Biotin; D012436:S-Adenosylmethionine; D000637:Transaminases; C102100:BioA protein, bacteria; D008239:Lysine