Identification,Biochemical Properties And Catalytic Application Of Glycoglycolipid Synthase From Marine Bacteria

The response and adaptation of marine microorganisms to environmental stresses are cutting-edge scientific issues in the field of environmental microbiology.It is reported that there is a phenomenon of cell membrane lipid remodeling under the condition of phosphorus limitation in the model strains of rhodobacteria and SAR11.Marine bacteria can cope with the low phosphorus environment of marine ecology by synthesizing a series of non phosphorus and nitrogen-containing membrane lipids and glycolipids instead of normal phospholipids.Therefore,the lipid metabolism and regulation mechanism of the cell membrane of marine bacteria under the condition of phosphorus limitation have become the research frontier and hotspot in this field.The PlcP,a phospholipase of heterotrophic bacteria Candidatus Pelagibacter sp.HTCC7211,has been studied in depth in the process of membrane lipid remodeling under phosphorus deficiency,but the glycosyltransferase,another key enzyme in membrane lipid remodeling,has not been studied.By using biochemical and bioinformatics methods,the biochemical properties and catalytic reaction mechanism of glycosyltransferase were extensively studied,and the specific functions of glycosyltransferase in the process of glycolipid synthesis of marine bacteria were clarified,which is of great significance for understanding the biological function of membrane lipid reconstruction of marine bacteria.In this paper,the nucleotide sequence of GT_cpp was obtained from GenBank,and the target gene was amplified by in vitro PCR technology.The target gene was digested with NdeI and Sal I,and the recombinant plasmid pET15b/GT_cpp was obtained and successfully expressed in Escherichia coli BL21.The target protein was dissolved by descaling agent to maintain a stable and uniform state.The enzyme was purified3.5 times by Ni-NTA affinity chromatography and Superdex-200 gel filtration,and the specific activity was 47.1 U/mg,and the yield was 17.1%.Molecular sieve experiments show that the glycosyltransferase GT_cpp exists as a dimer in an aqueous solution.The thin-layer chromatography and liquid chromatography-mass spectrometry were used to analyze the catalytic synthesis of purified enzyme GT_cp.The results showed that the glycosyltransferase GT_cpp can catalyze UDP-hexose?UDP-Glu,UDP-Gal?/UDP-glucuronic acid?UDP-Glc?and diacylglycerol?DAG?for the synthesis of different glyceroglycolipids.The enzymatic properties of the glycosyltransferase GT_cpp were studied in detail.The optimal reaction temperature and pH of GT_cpp were 35?and 8.5,respectively.GT_cpp could still maintain more than60%of the enzyme activity in a 1 mol/L NaCl solution.Mg²⁺,Ca²⁺and Mn²⁺could significantly increase GT_cpp activity,while Hg²⁺,Cd²⁺,Ni²⁺and Co²⁺completely inactivate the enzyme activity.Reducing agent?2-mercaptoethanol,DTT?could slightly increase GT_cpp activity,while SDS and urea strongly inhibited the activity of GT_cp.In the substrate specificity study of GT_cp,GT_cpp had the highest transglycosyl activity on the UDP-Glu sugar donor,and under the best conditions,the conversion rate of GT_cpp to MGlu-DAG reached 42.7%after reaction for 10 hours..The amino acid sequence of GT_cpp was analyzed by bioinformatics,and phylogenetic tree was established.It was found that GT_cpp and its homologues formed new evolutionary branches in GT4 family.Through homology modeling,the three-dimensional structure of GT_cpp was constructed,and amino acid homology comparison showed that GT_cpp has the typical characteristics of GT4 family:a His104-Asp256 catalytic activity center,two UDP-sugar binding domains of GRVAXEKN and DVFVFPSXTDTFG and a Gly-Rich domain.The construction of mutants D256A,H104A and D256E showed that conserved amino acid residues Asp256 and His104 play an important role in glycosyl transfer.By constructing predicted mutants for substrate binding sites?G19A,G82A,G85A,D190A,K195A,T257A,F258A,G259A,E264A?and analyzing mutant activities,the binding mechanism of GT_cpp to UDP-sugar donors was revealed.The results showed that the amino acid residues?Gly19,Gly82,Gly85,Arg190,Lys195,Thr257,Phe258,Gly259,Glu264?are involved in UDP-sugar donor binding.The enzyme activity of mutant T257S increased by 1.6 times,probably because there is more binding space between Ser residue and substrate,which facilitates the interaction between Ser residue and sugar donor.The above results indicate that GT_cpp has a sugar donor recognition mechanism similar to the reported GT-B family glycosyltransferases.By analyzing GT_cpp forming dimers in aqueous solution,it was found that the hydrophobic residue Leu56 is essential for GT_cpp dimer formation while mutant L56A exists as a monomer.