Abstract
The ST0452 protein from the thermophilic archaean Sulfolobus tokodaii has been identified as an enzyme with multiple sugar-1-phosphate nucleotidylyltransferase and amino-sugar-1-phosphate acetyltransferase (amino-sugar-1-P AcTase) activities. Analysis of the protein showed that in addition to glucosamine-1-phosphate (GlcN-1-P) AcTase activity, it possesses unique galactosamine-1-phosphate (GalN-1-P) AcTase activity not detected in any other proteins. Comparison of the crystal structures of the ST0452 protein and GlmU from Escherichia coli (EcGlmU), which possesses only GlcN-1-P AcTase activity, showed that the overall sequence identity between these two proteins is less than 25 %, but the amino acid residues predicted to comprise the catalytic center of EcGlmU are conserved in the ST0452 protein. To understand the molecular mechanism by which the ST0452 amino-sugar-1-P AcTase activity recognizes two independent substrates, several ST0452 substitution and truncation mutant proteins were constructed and analyzed. We found that His308 is essential for both GalN-1-P and GlcN-1-P AcTase activities, whereas Tyr311 and Asn331 are important only for the GalN-1-P AcTase activity. In addition, deletion of the C-terminal 5 or 11 residues showed that the 11-residue C-terminal region exerts a modest stimulatory effect on GalN-1-P AcTase activity but dramatically suppresses GlcN-1-P AcTase activity. This region also appears to make an important contribution to the thermostability of the entire ST0452 protein. Systematic deletions from the C-terminus also demonstrated that the C-terminal region with the β-helix structure has an important role mediating the trimerization of the ST0452 protein. This is the first report of an analysis of a thermostable archaeal enzyme exhibiting multiple amino-sugar-1-P AcTase activities.
Similar content being viewed by others
References
Bricnkkötter A, Klöß H, Alpert C-A, Lengeler JW (2000) Pathways for the utilization of N-acetyl-galactosamine and galactosamine in Escherichia coli. Mol Microbiol 37:125–135
Gehring AM, Lees WJ, Mindiola DJ, Walsh CT, Brown ED (1996) Acetyltransfer precedes uridylyltransfer in the formation of UDP-N-acetylglucosamine in separable active sites of the bifunctional GlmU protein of Escherichia coli. Biochemistry 35:579–585
Jagtap PK, Soni V, Vithani N, Jhingan GD, Bais VS, Nandicoori VK, Prakash B (2012) Substrate-bound crystal structures reveal features unique to Mycobacterium tuberculosis N-acetyl-glucosamine 1-phosphate uridyltransferase and a catalytic mechanism for acetyl transfer. J Biol Chem 287:39524–39537
Konopka JB (2012) N-acetylglucosamine functions in cell signaling. Scientifica (Cairo). doi:10.6064/2012/489208
Kotnik M, Anderluh PS, Prezelj A (2007) Development of novel inhibitors targeting intracellular steps of peptidoglycan biosynthesis. Curr Pharm Des 13:2283–2309
Leloir LF (1951) The enzymatic transformation of uridine diphosphate glucose into a galactose derivative. Arch Biochem Biophys 33:186–190
Olsen LR, Roderick SL (2001) Structure of the Escherichia coli GlmU pyrophosphorylase and acetyltransferase active sites. Biochemistry 40:1913–1921
Olsen LR, Vetting MW, Roderick SL (2007) Structure of the E. coli bifunctional GlmU acetyltransferase active site with substrates and products. Protein Sci 16:1230–1235
Raetz C (1993) Bacterial endotoxins: extraordinary lipids that activate eucaryotic signal transduction. J Bacteriol 175:5745
Ramakrishnan V, Teng Q, Adams MWW (1997) Characterization of UDP amino sugars as major phosphocompounds in the hyperthermophilic archaeon Pyrococcus furiosus. J Bacteriol 179:1505–1512
Riddles PW, Blakeley RL, Zerner B (1983) Reassessment of Ellman’s reagent. Methods Enzymol 91:49–60
Zhang Z, Tsujimura M, Akutsu J, Sasaki M, Tajima H, Kawarabayasi Y (2005) Identification of an extremely thermostable enzyme with dual sugar-1-phosphate nucleotidylyltransferase activities from an acidothermophilic archaeon, Sulfolobus tokodaii strain 7. J Biol Chem 280:9698–9705
Zhang Z, Akutsu J, Tsujimura M, Kawarabayasi Y (2007) Increasing in Archaeal GlcNAc-1-P uridyltransferase activity by targeted mutagenesis while retaining its extreme thermostability. J Biochem 141:553–562
Zhang Z, Akutsu J, Kawarabayasi Y (2010) Identification of novel acetyltransferase activity on the thermostable protein ST0452 from Sulfolobus tokodaii strain 7. J Bacteriol 192:3287–3293
Acknowledgments
This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. It was also supported in part by the Institute for Fermentation, Osaka (IFO).
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by H. Atomi.
Rights and permissions
About this article
Cite this article
Zhang, Z., Shimizu, Y. & Kawarabayasi, Y. Characterization of the amino acid residues mediating the unique amino-sugar-1-phosphate acetyltransferase activity of the archaeal ST0452 protein. Extremophiles 19, 417–427 (2015). https://doi.org/10.1007/s00792-014-0727-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00792-014-0727-9