Heterelogous Expression And Functional Roles Of A Udp-Glycosyltransferase GFUGT88A1 In Grifola Frondosa

UDP-glycosyltransferases（Uridine Diphosphate-Glycosyltransferases,UGTs）widely exist in animals,plants,fungi and bacteria,and play many critical roles in the glycosylation of plant natural products,antibiotic synthesis,development of bacterial glycosyltransferase inhibitors,and investigation of inflammation,hematopoiesis,development,and the nervous system in animals.To date,functions and catalytic mechanisms of UGTs from plants,bacteria,and animals have been extensively studied.However,data on fungal UGTs and their roles are rarely available.Therefore,the present study cloned and sequenced the gene gfugt88a1 from Grifola frondosa encoding an UDP-glycosyltransferases 88A1（GFUGT88A1）,carried out the heterologous expression of GFUGT88A1,analyzed its potential catalytic mechanism,and predicted the three-dimensional（3D）conformation and molecular docking sites.Our findings provide a reference to better understand the catalytic mechanism of other UDP-glucosyltransferase.The main results and conclusions are obtained as follows:（1）A UDP-glucosyltransferase gene gfugt88a1 from G.frondosa was cloned,sequenced and bioinformatics analyzed.The full-length g DNA sequence is 1951 bp,and the full-length c DNA sequence is 1389 bp,encoding mature polypeptide with a total of 462 amino acids,a Mw of 50.96 k Da and p I of 5.29.GFUGT88A1 was predicted as a non-secretory protein with no transmembrane structures and signal peptides.GFUGT88A1p belonged to the GT1 family,adopted GT-B fold type and contained 4 typical conserved domains and 6 TDP-binding sites.Combined with the phylogenetic tree and multiple sequence alignment information,GFUGT88A1 was classed into a branch with seven UGTs of the O.rivulosa,and the homology between similar sequences from different basidiomycetes was low（～50%）,but their C-terminal conserved domain maintained high similarity andcontained a typical highly conserved characteristic sequence.（2）G.frondosa UDP-glucosyltransferase GFUGT88A1 was heterelogously expressed,purified and enzymatically characterizied.By codon-optimizing,constructing the expression plasmid p ET-30a（+）-gfugt88A1 and IPTG induction at low temperature,the recombinant GFUGT88A1 was obtained and further purified with yield of 16.05%and the specific activity of53.02 U·μg^-1 after ultrasonic disruption,denaturation treatment,affinity chromatography,dialysis,and ultrafiltration concentration.The Mw of GFUGT88A1 was predicted as approximately 51.7k Da by trypsin treatment,liquid chromatography-MS/MS and Mascot search analysis,matching the expected Mws of the cloned GFUGT88A1（51 k Da）.GFUGT88A1 showed a broad substrate specificity for small molecule compounds and mono/oligosaccharides（DP=1,2,6,9）.GFUGT88A1 showed an extremely low UDP-glucosyltransferase activity when glucose was used as the substrate.Enzyme kinetic analysis of GFUGT88A1 using UDP-glucose as donor and oligosaccharides as acceptor showed that GFUGT88A1 had the highest affinity for LAM-9 with a Km value of 24.15±0.56μM,and the highest catalytic efficiency with a Kcat/Km value of0.055±0.011 min^-1μM^-1,indicating that LAM-9 is the preferred acceptor for GFUGT88A1.Enzymatic properties showed that GFUGT88A1 had an optimum temperature of 37°C and p H of7.0,showing high thermal stability and p H stability.Metal ions Fe³⁺,Fe²⁺,Cu²⁺,and Zn²⁺have a strong inhibitory effect on the activity of GFUGT88A1 with the inhibition rate of less than 40%,While Na⁺increased GFUGT88A1 activity by approximately 20%.（3）The potential catalytic mechanism of UDP-glucosyltransferase GFUGT88A1 was analyzed based on the synthesized products,the three-dimensional（3D）conformation,and molecular docking sites.The GFUGT88A1 showed a broad substrate specificity for all oligosaccharides LAM2,LAM6,and LAM9 as acceptor using UDP-glucose as donor.The peaks representing donor UDP-glc and acceptor LAM2 at a retention time of 1.89 min and/or 4.20 min were observed in standard solutions,and control and reaction groups,respectively.A peak at a retention time of 5.25 min was identified as a synthesized oligosaccharide with a DP of 4calculated using the aforementioned standard equation.Accordingly,at least two peaks representing synthesized oligosaccharides with a DP of 8 were obtained when using LAM6 as an acceptor.When LAM9 was used as an acceptor,a synthesized oligosaccharide with a DP of 15was identified with a retention time of 10.99 min.Molecular modeling revealed that the lobes in GFUGT88A1 had a typical Rossmann-like fold（β/α/β）and were packed tightly to form a deep cleft serving as the binding site of the sugar acceptor substrate.The stereochemical parameters of the GFUGT88A1 model were evaluated using the SAVES and Pro SA,which indicated that the GFUGT88A1 model was reliable and of a good quality.Docking and active site analysis results proved that Trp323 and Glu349 play a key role in the recognition and binding of UDP-Glc as a donor,and the sugar moiety of the acceptor and positioning direction of the glycosylation reaction,respectively.