Complementation of an E. coli cysteine auxotrophic mutant for the structural modification study of 3'(2'),5'-bisphosphate nucleotidase.
The Arabidopsis AHL gene encodes a 3'(2'),5'-bisphosphate nucleotidase (BPNTase) involved in the reductive sulfate activation pathway. A bacterial expression vector containing AHL cDNA was randomly mutagenized with hydroxylamine and transformed into the E. coli cysteine auxotrophic mutant cysQ. Bacterial colonies that did not show evidence of complementation, i.e. those that exhibited slower growth on cysteine-free medium, were selected for further study. Sequencing of the AHL cDNA in one such clone revealed the conversion of cytosine 635 (C635) to thymine, resulting in an Alanine (A212) to Valine substitution. This microbial complementation procedure is useful in BPNTase structure-activity studies for biotechnological applications.
Cheong
Jong-Joo
JJ
School of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University, Seoul, Korea. cheongjj@snu.ac.kr
Hwang
Ingyu
I
Rhee
Sangkee
S
Moon
Tae Wha
TW
Choi
Yang Do
YD
Kwon
Hawk-Bin
HB
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D003596:Cytosine / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D009708:Nucleotidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013941:Thymine / Q000737:chemistry / Q000378:metabolism; D014633:Valine / Q000235:genetics
D029681:Arabidopsis Proteins; D003596:Cytosine; C510507:CysQ protein, E coli; D009708:Nucleotidases; D010744:Phosphoric Monoester Hydrolases; C053354:bisphosphoadenylate 3'-nucleotidase; D014633:Valine; D003545:Cysteine; D000409:Alanine; D013941:Thymine