Variation of cofactor levels in Escherichia coli. Sequence analysis and expression of the pncB gene encoding nicotinic acid phosphoribosyltransferase.
The pncB gene from Escherichia coli, which encodes nicotinic acid phosphoribosyltransferase (EC 2.4.2.11), was cloned on a 1.5-kilobase TaqI-EcoRI fragment. Its position on the E. coli chromosome was determined at 20.8 min between the asnS and pepN loci. The nucleotide sequence of the gene and the transcription and translation initiation sites were determined. Expression of pncB on a multicopy plasmid leads to a 25-fold increase in nicotinic acid phosphoribosyltransferase activity. Growth of E. coli in the presence of nicotinic acid leads to strong repression of nicotinic acid phosphoribosyltransferase activity, indicating that the cloned pncB sequence contains its own control sequences. It is shown that increased nicotinic acid phosphoribosyltransferase activity effects a 5-fold increase in the intracellular concentration of NAD. The cloned pncB gene can therefore be used as a tool to raise intracellular cofactor levels.
Wubbolts
M G
MG
Department of Chemistry, University of Groningen, The Netherlands.
Terpstra
P
P
van Beilen
J B
JB
Kingma
J
J
Meesters
H A
HA
Witholt
B
B
D000595:Amino Acid Sequence; D001483:Base Sequence; D002876:Chromosomes, Bacterial; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial; D005838:Genotype; D007700:Kinetics; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism*; D010430:Pentosyltransferases / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D009243:NAD; D010430:Pentosyltransferases; C021864:nicotinate phosphoribosyltransferase