A Chimeric UDP-Glucose Pyrophosphorylase Produced by Protein Engineering Exhibits Sensitivity to Allosteric Regulators

International Journal of Molecular Sciences

Mat໚s D. Asención Diez²  Ana C. Ebrecht²  Lucila I. Martínez²  Mabel C. Aleanzi²  Sergio A. Guerrero²  Miguel A. Ballໜora¹ 

[1] Department of Chemistry and Biochemistry, Loyola University Chicago, 1068 W Sheridan Rd., Chicago, IL 60660, USA; E-Mail:;Instituto de Agrobiotecnología del Litoral (UNL-CONICET), Facultad de Bioquímica y Ciencias Biológicas, Paraje “El Pozo” CC 242, S3000ZAA Santa Fe, Argentina; E-Mails:

关键词: protein engineering;    allosteric regulation;    pyrophosphorylases evolution;    UDP-glucose;    ADP-glucose;   

DOI  :  10.3390/ijms14059703

来源: mdpi

【 摘 要 】

In bacteria, glycogen or oligosaccharide accumulation involves glucose-1-phosphate partitioning into either ADP-glucose (ADP-Glc) or UDP-Glc. Their respective synthesis is catalyzed by allosterically regulated ADP-Glc pyrophosphorylase (EC 2.7.7.27, ADP-Glc PPase) or unregulated UDP-Glc PPase (EC 2.7.7.9). In this work, we characterized the UDP-Glc PPase from Streptococcus mutans. In addition, we constructed a chimeric protein by cutting the C-terminal domain of the ADP-Glc PPase from Escherichia coli and pasting it to the entire S. mutans UDP-Glc PPase. Both proteins were fully active as UDP-Glc PPases and their kinetic parameters were measured. The chimeric enzyme had a slightly higher affinity for substrates than the native S. mutans UDP-Glc PPase, but the maximal activity was four times lower. Interestingly, the chimeric protein was sensitive to regulation by pyruvate, 3-phosphoglyceric acid and fructose-1,6-bis-phosphate, which are known to be effectors of ADP-Glc PPases from different sources. The three compounds activated the chimeric enzyme up to three-fold, and increased the affinity for substrates. This chimeric protein is the first reported UDP-Glc PPase with allosteric regulatory properties. In addition, this is a pioneer work dealing with a chimeric enzyme constructed as a hybrid of two pyrophosphorylases with different specificity toward nucleoside-diphospho-glucose and our results turn to be relevant for a deeper understanding of the evolution of allosterism in this family of enzymes.

【 授权许可】

CC BY   
© 2013 by the authors; licensee MDPI, Basel, Switzerland