SanJ, an ATP-dependent picolinate-CoA ligase, catalyzes the conversion of picolinate to picolinate-CoA during nikkomycin biosynthesis in Streptomyces ansochromogenes.
Nikkomycins, a group of peptidyl nucleoside antibiotics, are competitive inhibitors of chitin synthase. The nikkomycin biosynthetic gene cluster has been cloned previously from Streptomyces ansochromogenes. The cluster contains 25 complete ORFs including sanJ. The sanJ gene was inactivated by the insertion of a kanamycin resistance gene and the resulting disruption mutants failed to produce nikkomycins. Moreover, the nikkomycin production was restored by complementation with a single copy of sanJ. The deduced product of sanJ bears striking sequence similarity with enzymes belonging to the adenylate-forming superfamily. sanJ was overexpressed as a His6-tagged fusion protein in Escherichia coli and purified to apparent homogeneity by affinity chromatography. The purified SanJ demonstrated adenylate ligase activity in the presence of picolinate or its analogs (benzoate, nicotinate, 4-methoxybenzoate, 4-hydroxybenzoate), ATP and Mg2+. SanJ was also found to catalyze the conversion of picolinate, benzoate, nicotinate to their corresponding CoA esters and 4-methoxybenzoate, 4-hydroxybenzoate to their respective AMP derivatives in vitro. This was unambiguously shown by using HPLC and electrospray ionization mass spectrometry (ESI-MS) or by comparing the reaction product with an authentic standard of benzoyl-CoA. These results indicated that sanJ encodes an ATP-dependent picolinate-CoA ligase which is essential for nikkomycin biosynthesis.
Niu
Guoqing
G
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China.
Liu
Gang
G
Tian
Yuqing
Y
Tan
Huarong
H
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000617:Aminoglycosides / Q000096:biosynthesis*; D002384:Catalysis; D003066:Coenzyme A Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D010848:Picolinic Acids / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013302:Streptomyces / Q000378:metabolism*; D013379:Substrate Specificity
D000617:Aminoglycosides; D010848:Picolinic Acids; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; C011952:nikkomycin; D003066:Coenzyme A Ligases; C512544:SanJ protein, Streptomyces ansochromogenes; C030614:picolinic acid