A Glycolipid Glycosyltransferase with Broad Substrate Specificity from the Marine Bacterium “ Candidatus Pelagibacter sp.” Strain HTCC7211

Abstract

In the marine environment, phosphorus availability significantly affects the lipid composition in many cosmopolitan marine heterotrophic bacteria, including members of the SAR11 clade and the Roseobacter clade. Under phosphorus stress conditions, non-phosphorus sugar-containing glycoglycerolipids are substitutes for phospholipids in these bacteria. Although these glycoglycerolipids play an important role as surrogates for phospholipids under phosphate deprivation, glycoglycerolipid synthases in marine microbes are poorly studied. In the present study, we biochemically characterized a glycolipid glycosyltransferase (GTcp) from the marine bacterium Candidatus Pelagibacter sp. HTCC7211, a member of the SAR11 clade. Our results showed that GTcp is able to act as a multifunctional enzyme by synthesizing different glycoglycerolipids with UDP-glucose, UDP-galactose, or UDP-glucuronic acid as sugar donors and diacylglycerol as the acceptor. Analyses of enzyme kinetic parameters demonstrated that Mg2+ notably changes the enzyme’s affinity for UDP-glucose, which improves its catalytic efficiency. Homology modelling and mutational analyses revealed binding sites for the sugar donor and the diacylglycerol lipid acceptor, which provided insights into the retaining mechanism of GTcp with its GT-B fold. A phylogenetic analysis showed that GTcp and its homologs form a group in the GT4 glycosyltransferase family. These results not only provide new insights into the glycoglycerolipid synthesis mechanism in lipid remodelling, but also describe an efficient enzymatic tool for future synthesis of bioactive molecules. Importance The bilayer formed by membrane lipids serves as the containment unit for living microbial cells. In the marine environment, it has been firmly established that phytoplankton and heterotrophic bacteria can substitute phospholipids with non-phosphorus sugar-containing glycoglycerolipids in response to phosphorus limitation. However, little is known about how these glycoglycerolipids are synthesized. Here, we determined the biochemical characteristics of a glycolipid glycosyltransferase (GTcp) from the marine bacterium Candidatus Pelagibacter sp. HTCC7211. GTcp and its homologs form a group in the GT4 glycosyltransferase family, and can synthesize neutral glycolipids (MGlc-DAG and MGal-DAG) and an acidic glycolipid (MGlcA-DAG). We also uncover the key residues for DAG-binding through molecular docking, site-direct mutagenesis and subsequent enzyme activity assays. Our data provide new insights into the glycoglycerolipid synthesis mechanism in lipid remodelling.

The Proteobacterial Methanotroph Methylosinus trichosporium OB3b Remodels Membrane Lipids in Response to Phosphate Limitation

“…3 ). PlcP and Agt of strain OB3b had 51 and 43% sequence identity to the characterized counterparts from Pelagibacter ubique HTCC7211, respectively ( 22 , 23 ). Multiple sequence alignment and phylogenetic analyses of PlcP ( Fig.…”

“…LpxH encoding an enzyme involved in lipid A biosynthesis is used as an outgroup ( 49 , 50 ). (E) Neighbor-joining phylogenetic tree showing Agt of strain OB3b, together with characterized GT4-group glycosyltransferases, including Agt from Pelagibacter ubique ( 19 , 23 ), Agrobacterium tumefaciens ( 21 ), and Pseudomonas aeruginosa ( 17 ). MshA involved in mycothiol biosynthesis is used as an outgroup ( 51 , 52 ).…”