Substrate specificities of family 1 UGTs gained by domain swapping

• Published in: Phytochemistry (Oxford) Vol. 70; no. 4; pp. 473 - 482
• Main Authors: Hansen, Esben Halkjær, Osmani, Sarah A., Kristensen, Charlotte, Møller, Birger Lindberg, Hansen, Jørgen
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier Ltd 01.03.2009; Elsevier
• Subjects: ADME improvement; Arabidopsis - enzymology; Arabidopsis - genetics; Asteraceae - chemistry; Asteraceae - enzymology; Biocatalysis; Biological and medical sciences; Chemical constitution; Combinatorial biochemistry; Curcumin; Domain swapping; Etoposide; Fundamental and applied biological sciences. Psychology; Glycosylation; Glycosyltransferase; Glycosyltransferases - chemistry; Glycosyltransferases - genetics; Glycosyltransferases - physiology; Kinetics; Models, Molecular; Plant physiology and development; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - physiology; Protein Engineering; Protein Structure, Tertiary; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - physiology; Resveratrol; Sorghum - chemistry; Sorghum - enzymology; Stevia - enzymology; Stevia - genetics; Substrate Specificity; Biocatalysis; Glycosyltransferase; Domain swapping; ADME improvement; Resveratrol; Etoposide; Combinatorial biochemistry; Glycosylation; Curcumin; Antineoplastic agent; Glycosyltransferases; Podophyllotoxin derivatives; Isomerases; Substrate specificity; Antimitotic; DNA topoisomerase (ATP-hydrolysing); UDP-glucuronosyltransferase; Enzyme; Tyrosine kinase inhibitor; Transferases; Chemical structure; Enzyme inhibitor; Topoisomerase II inhibitor; Antioxidant; Stilbene derivatives; Plant origin; Hexosyltransferases
• ISSN: 0031-9422; 1873-3700
• DOI: 10.1016/j.phytochem.2009.01.013

Chimeric family 1 UDP-glucose glycosyltransferases (UGTs) with substrates specificities were constructed. Twelve active chimeras were obtained, using domains of the UGTs 71C1, 71C2, 71E1 and 85C1, of two different plant species. Among activities observed were regio-specific glycosylation of resveratrol and increased efficiency of etoposide glycosylation. Family 1 glycosyltransferases are a group of enzymes known to embrace a large range of different substrates. This study devises a method to enhance the range of substrates even further by combining domains from different glycosyltransferases to gain improved substrate specificity and catalytic efficiency. Chimeric glycosyltransferases were made by combining domains from seven different family 1 glycosyltransferases, UGT71C1, UGT71C2, UGT71E1, UGT85C1, UGT85B1, UGT88B1 and UGT94B1. Twenty different chimeric glycosyltransferases were formed of which twelve were shown to be catalytically active. The chimeric enzymes of Arabidopsis thaliana UGT71C1 and UGT71C2 showed major changes in acceptor substrate specificity and were able to glycosylate etoposide significantly better than the parental UGT71C1 and UGT71C2 enzymes, with K cat and efficiency coefficients 3.0 and 2.6 times higher, respectively. Chimeric glycosyltransferases of UGT71C1 combined with Stevia rebaudiana UGT71E1, also afforded enzymes with high catalytic efficiency, even though the two enzymes only display 38% amino acid sequence identity. These chimeras show a significantly altered regiospecificity towards especially trans-resveratrol, enabling the production of trans-resveratrol-β-4′- O-glucoside (resveratroloside). The study demonstrates that it is possible to obtain improved catalytic properties by combining domains from both closely as well as more distantly related glycosyltransferases. The substrate specificity gained by the chimeras is difficult to predict because factors determining the acceptor specificity reside in the N- terminal as well as the C-terminal domains.