Identification and characterization of UDP-glucose pyrophosphorylase in cyanobacteria Anabaena sp. PCC 7120.
Exopolysaccharides produced by photosynthetic cyanobacteria have received considerable attention in recent years for their potential applications in the production of renewable biofuels. Particularly, cyanobacterial cellulose is one of the most promising products because it is extracellularly secreted as a non-crystalline form, which can be easily harvested from the media and converted into glucose units. In cyanobacteria, the production of UDP-glucose, the cellulose precursor, is a key step in the cellulose synthesis pathway. UDP-glucose is synthesized from UTP and glucose-1-phosphate (Glc-1P) by UDP-glucose pyrophosphorylase (UGPase), but this pathway in cyanobacteria has not been well characterized. Therefore, to elucidate the overall cellulose biosynthesis pathway in cyanobacteria, we studied the putative UGPase All3274 and seven other putative NDP-sugar pyrophosphorylases (NSPases), All4645, Alr2825, Alr4491, Alr0188, Alr3400, Alr2361, and Alr3921 of Anabaena sp. PCC 7120. Assays using the purified recombinant proteins revealed that All3274 exhibited UGPase activity, All4645, Alr2825, Alr4491, Alr0188, and Alr3921 exhibited pyrophosphorylase activities on ADP-glucose, CDP-glucose, dTDP-glucose, GDP-mannose, and UDP-N-acetylglucosamine, respectively. Further characterization of All3274 revealed that the kcat for UDP-glucose formation was one or two orders lower than those of other known UGPases. The activity and dimerization tendency of All3274 increased at higher enzyme concentrations, implying catalytic activation by dimerization. However, most interestingly, All3274 dimerization was inhibited by UTP and Glc-1P, but not by UDP-glucose. This study presents the first in vitro characterization of a cyanobacterial UGPase, and provides insights into biotechnological attempts to utilize the photosynthetic production of cellulose from cyanobacteria.
Faculty of Agriculture, Shinshu University, 8304 Minamiminowa, Nagano 399-4511, Japan; Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
Yusuke
Y
Kawano
Faculty of Agriculture, Shinshu University, 8304 Minamiminowa, Nagano 399-4511, Japan.
Midori
M
Sekine
Faculty of Agriculture, Shinshu University, 8304 Minamiminowa, Nagano 399-4511, Japan; JST, PRESTO, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan. Electronic address: m_ihara@shinshu-u.ac.jp.
Masaki
M
Ihara
D000245:Adenosine Diphosphate Glucose / Q000378:metabolism; D000595:Amino Acid Sequence; D017033:Anabaena / Q000201:enzymology*; D002482:Cellulose / Q000378:metabolism; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D005947:Glucose / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009702:Nucleoside Diphosphate Sugars / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013942:Thymine Nucleotides / Q000378:metabolism; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D003432:Cross-Linking Reagents; D009702:Nucleoside Diphosphate Sugars; D011994:Recombinant Proteins; D013942:Thymine Nucleotides; C073881:cytidine diphosphate-glucose; C103522:deoxythymidine diphosphate-glucose; D000245:Adenosine Diphosphate Glucose; D002482:Cellulose; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase; D005947:Glucose
Anabaena sp. PCC 7120; Cellulose; Cyanobacteria; Nucleotidyltransferase; UDP-glucose; UDP-glucose pyrophosphorylase