Sequence Properties of the 1,2-Diacylglycerol 3-Glucosyltransferase from Acholeplasma laidlawiiMembranes

Synthesis of the nonbilayer-prone α-monoglucosyldiacylglycerol (MGlcDAG) is crucial for bilayer packing properties and the lipid surface charge density in the membrane ofAcholeplasma laidlawii. The gene for the responsible, membrane-bound glucosyltransferase (alMGS) (EC 2.4.1.157) was sequenced and functionally cloned in Escherichia coli, yielding MGlcDAG in the recombinants. Similar amino acid sequences were encoded in the genomes of several Gram-positive bacteria (especially pathogens), thermophiles, archaea, and a few eukaryotes. All of these contained the typical EX7E catalytic motif of the CAZy family 4 of α-glycosyltransferases. The synthesis of MGlcDAG by a close sequence analog from Streptococcus pneumoniae (spMGS) was verified by polymerase chain reaction cloning, corroborating a connection between sequence and functional similarity for these proteins. However, alMGS and spMGS varied in dependence on anionic phospholipid activators phosphatidylglycerol and cardiolipin, suggesting certain regulatory differences. Fold predictions strongly indicated a similarity for alMGS (and spMGS) with the two-domain structure of the E. coli MurG cell envelope glycosyltransferase and several amphipathic membrane-binding segments in various proteins. On the basis of this structure, the alMGS sequence charge distribution, and anionic phospholipid dependence, a model for the bilayer surface binding and activity is proposed for this regulatory enzyme. Synthesis of the nonbilayer-prone α-monoglucosyldiacylglycerol (MGlcDAG) is crucial for bilayer packing properties and the lipid surface charge density in the membrane ofAcholeplasma laidlawii. The gene for the responsible, membrane-bound glucosyltransferase (alMGS) (EC 2.4.1.157) was sequenced and functionally cloned in Escherichia coli, yielding MGlcDAG in the recombinants. Similar amino acid sequences were encoded in the genomes of several Gram-positive bacteria (especially pathogens), thermophiles, archaea, and a few eukaryotes. All of these contained the typical EX7E catalytic motif of the CAZy family 4 of α-glycosyltransferases. The synthesis of MGlcDAG by a close sequence analog from Streptococcus pneumoniae (spMGS) was verified by polymerase chain reaction cloning, corroborating a connection between sequence and functional similarity for these proteins. However, alMGS and spMGS varied in dependence on anionic phospholipid activators phosphatidylglycerol and cardiolipin, suggesting certain regulatory differences. Fold predictions strongly indicated a similarity for alMGS (and spMGS) with the two-domain structure of the E. coli MurG cell envelope glycosyltransferase and several amphipathic membrane-binding segments in various proteins. On the basis of this structure, the alMGS sequence charge distribution, and anionic phospholipid dependence, a model for the bilayer surface binding and activity is proposed for this regulatory enzyme. 1,2-diacyl-3-O-(α-d-glucopyranosyl)-sn-glycerol A. laidlawii MGlcDAG synthase 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid cardiolipin 2-DAG, 1,2-diacylglycerol 1,2-diacyl-3-O-[α-d-glucopyranosyl-(1→2)-O-α-d-glucopyranosyl]-sn-glycerol glycosyltransferase 1,2-diacyl-3-O-(β-d-galactopyranosyl)-sn-glycerol phosphatidylethanolamine phosphatidylglycerol dodecylphosphate-rac-glycerol S. pneumoniae MGlcDAG synthase polymerase chain reaction MGlcDAG synthase transmembrane group of overlapping clones glucose Lipids are the local environment for most integral and peripheral membrane proteins, which often depend on the lipids for optimal function. The large diversity of lipids and the differences in composition and properties between membranes have made it difficult to find out common features of bilayer organization and how lipids and proteins are cooperating in local processes. Lipid-synthesizing pathways have been mapped for the most common types of lipids, and several of the corresponding enzymes catalyzing these reactions have been characterized. However, when it comes to the connection between regulation of bilayer properties and enzyme structure, very little is known (1Hjelmstad R. Bell R. Biochemistry. 1991; 30: 1731-1740Crossref PubMed Scopus (52) Google Scholar). So far, only a few lipid-synthesizing enzymes have been crystallized. Which structural properties are involved in the catalytic mechanism of these lipid enzymes, and how are the membrane properties sensed (1Hjelmstad R. Bell R. Biochemistry. 1991; 30: 1731-1740Crossref PubMed Scopus (52) Google Scholar)? In the well characterized plasma membrane of Acholeplasma laidlawii, the lipid composition is regulated in a manner to maintain (i) lipid phase equilibria, close to a potential bilayer to nonbilayer transition, (ii) a nearly constant radius of spontaneous curvature, and (iii) a certain anionic surface charge density of the lipid bilayer. The synthesis of the major nonbilayer-prone lipid in this membrane, monoglucosyldiacylglycerol (MGlcDAG)1 (Scheme FS1, step I), plays an important role to fulfill the two first points above but also the third, since it is strongly regulated by negatively charged lipids (e.g. the major in vivo lipid phosphatidylglycerol (PG)) (2Karlsson O.P. Dahlqvist A. Wieslander Å. J. Biol. Chem. 1994; 269: 23484-23490Abstract Full Text PDF PubMed Google Scholar). MGlcDAG is consecutively processed into diglucosyl diacylglycerol (DGlcDAG) (Scheme FS1, step II). Consequently, the formation of this glucolipid, a transfer of Glc from the donor UDP-Glc to the acceptor lipid diacylglycerol (DAG) catalyzed by a glucosyltransferase (EC 2.4.1.157) (3Karlsson O.P. Dahlqvist A. Vikstroem S. Wieslander A. J. Biol. Chem. 1997; 272: 929-936Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), plays a central part in understanding the total regulation of lipid syntheses in A. laidlawii membranes. Furthermore, glycolipids including nonbilayer-prone ones are major constituents in many cell surface membranes, certain bacterial groups, and most photosynthetic organelles. Fairly little is known about the synthesis and regulation of these. Usually, they are made in a separate pathway (as in A. laidlawii), branching from the conserved one to anionic phospholipids. In this work, we have cloned the gene for the α-monoglucosyldiacylglycerol synthase from A. laidlawiimembranes and a sequence analog from the pathogen Streptococcus pneumoniae and propose these genes, on the basis of sequence similarities, to belong to a new large group of lipid glycosyltransferases that are widely spread in nature. We also present a functional comparison between the two cloned glucosyltransferases and discuss structural properties based on two- and three-dimensional fold predictions from the primary structure. A striking similarity to two new, related structures for an Escherichia coliglycosyltransferase and epimerase, respectively, is indicated. Strain A-EF22 of A. laidlawii was cultivated as described by Karlsson et al. (3Karlsson O.P. Dahlqvist A. Vikstroem S. Wieslander A. J. Biol. Chem. 1997; 272: 929-936Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), and genomic DNA was prepared using the kit GenomicPrepTM (Amersham Pharmacia Biotech). The growth ofS. pneumoniae strain 19F CCUG 3030 was performed in Todd-Hewitt medium. For DNA extraction, an overnight culture was harvested, and the pellet was resuspended in H2O and heated at 95 °C for 10 min. The supernatant from the following centrifugation contained the DNA. The N-terminal sequence of the purified MGlcDAG synthase from A. laidlawii (alMGS) (3Karlsson O.P. Dahlqvist A. Vikstroem S. Wieslander A. J. Biol. Chem. 1997; 272: 929-936Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) was analyzed through Edman degradation, revealing a 20-residue sequence (4Karlsson O.P. Maintenance of Lipid bilayer properties: Key role of two glucolipid synthases in the membranes of Acholeplasma kidlawii . Ph.D. thesis. Department of Biochemistry , Umeå University, Umeå, Sweden1997Google Scholar). An internal amino acid sequence of 10 residues was determined as above after proteolytic cleavage of the protein and separation of peptides by reverse phase high pressure liquid chromatography. Degenerated oligonucleotides with the primary sequence 5′-ATT GGT ATI TT(T/C) TCI GAA GC-3′ and 5′-TTT ATC TGG were and in a with genomic A. laidlawii DNA as the using DNA polymerase were at °C for °C for and °C for by a at °C for 10 min. was into a and cloned in for was performed by with DNA of clones was performed using and was for a of A. laidlawii genomic DNA. The DNA were by and to a membrane in a R. in Scholar). by with from the cloned of the gene were by and in DNA for at DNA with the corresponding to the were purified and in a reaction with the by membrane, and the kit were from Pharmacia The was by were for the and for the gene from S. pneumoniae with the structure and sequence The corresponding oligonucleotides for the gene from A. were with the sequence GGT and DNA was for and the were into the and cloned as described The alMGS was also with an N-terminal S. and A. for using the E. coli strain for of the and were on with of was performed in with of The were at and was at were by centrifugation after of was from and cardiolipin were from Lipids The MGlcDAG was prepared as described by al. (3Karlsson O.P. Dahlqvist A. Vikstroem S. Wieslander A. J. Biol. Chem. 1997; 272: 929-936Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). in a with the cloned MGlcDAG was from the with was from and was from E. coli were in and by on for The protein in cell were between and determined by a kit were prepared as described by Karlsson et al. (3Karlsson O.P. Dahlqvist A. Vikstroem S. Wieslander A. J. Biol. Chem. 1997; 272: 929-936Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), with an for the In the for MGlcDAG of protein was to of lipid and on for min. The reaction was by the of of to a of lipid was 10 in to the of at the reaction was with of and the lipids were and by (2Karlsson O.P. Dahlqvist A. Wieslander Å. J. Biol. Chem. 1994; 269: 23484-23490Abstract Full Text PDF PubMed Google Scholar). from MGlcDAG synthesis in vivo was in a synthesis S. Karlsson O.P. Wieslander Å. Biochemistry. PubMed Scopus Google Scholar), using a nearly of the synthase from A. laidlawii. The lipid on the were and by All were in The glucolipid were also with a Lipid were first by as with a and to Lipids were after Lipid An and Scholar). were in of and for to the of The were at and was at were by centrifugation after 4 of Lipids were from the cell pellet by and and by in acid were by The is into the lipid and was to for cardiolipin which was to A in was also performed with of of The amino acid sequence of the alMGS was in for sequences with J. 1997; PubMed Scopus Google Scholar) at the in the of and and the at sequence were from the for through in and are from of the primary and structures of the sequences were performed with at the of and with the For the three-dimensional structure the three-dimensional fold at the S. J. Biol. PubMed Scopus Google Scholar) and J. PubMed Scopus Google Scholar) at were from the for the MGlcDAG synthase with are are in and with high amino acid All are determined to glycosyltransferases of various and of CAZy family in a new from the for the MGlcDAG synthase with are are in and with high amino acid All are determined to glycosyltransferases of various and of CAZy family sequence have been at with the purified MGlcDAG synthase of A. laidlawii (3Karlsson O.P. Dahlqvist A. Vikstroem S. Wieslander A. J. Biol. Chem. 1997; 272: 929-936Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), the N-terminal and an internal amino acid sequence was A from DNA with the corresponding DNA a was as a for in a and a sequence described in An of a at and a potential were on the basis of a of the sequence with the two amino acid sequences The of the DNA was typical for was for a protein with amino a to and the gene The amino acid sequence is related to the ones in the A. A. S. J. J. J. 1997; PubMed Scopus Google Scholar). potential lipid-synthesizing enzyme were present on the However, the two acid synthases a conserved The A. been described in R. A. 1991; PubMed Scopus Google of the of to MGlcDAG synthase in A. to to to to A. in a new The amino acid sequence for was as a in a A of the are in the ones glycosyltransferases from a large of were All of these belong to family 4 in the glycosyltransferase J. J. 1997; PubMed Scopus Google Scholar) and the typical residues for in this and belong to family in the by et al. E. A. J. PubMed Scopus Google Scholar). However, the were in a for and the was a sequence for a protein in with amino acid to the MGlcDAG synthase above The sequences from S. were with the alMGS sequence The conserved residues in sequences were to the first residues and above amino which the motif EX7E of family 4 A in the protein that alMGS and the potential to which is the glycosyltransferase group proteins transfer and to a of acceptor as and of the S. pneumoniae gene been but with J. R. J. 1997; PubMed Google the sequence was from a in the and and was the In to the potential of the proteins in in the was pneumoniae DNA. The were of the but out of with the gene in the in an E. coli A at from the was for The was also into a and coli as a protein with a to the from and were by and in for MGlcDAG synthesis were using and lipid to O.P. Dahlqvist A. Vikstroem S. Wieslander A. J. Biol. Chem. 1997; 272: 929-936Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The that the encoded proteins were to the The was of which of the two was In to that the lipid in vivo was it was from the and in an in for synthesis with purified synthase from A. laidlawii membranes S. Karlsson O.P. Wieslander Å. Biochemistry. PubMed Scopus Google Scholar). enzyme only as the lipid and MGlcDAG The lipid as in the synthesis The of the was when was The MGlcDAG was also as a by with A typical for glycolipids was lipids in E. coli in and in vivo lipid in a new the two and with the amino acid sequences indicated in enzymes, which the synthesis of the membrane lipid The lipid composition in the E. coli was analyzed by of into the lipids major lipids were on the the glucolipid phosphatidylethanolamine and the negatively charged lipids phosphatidylglycerol and The a a lipid composition for E. coli with about and negatively charged lipids The strain with alMGS contained a of about The of anionic lipids was the nonbilayer lipid to about However, the glucosyltransferase from S. in the lipid and only of MGlcDAG were The the a synthesis of the glucolipid with that the N-terminal enzyme The the growth with and the the phase which the of and The A. laidlawii MGlcDAG synthase is in an manner by of negatively charged lipids, (2Karlsson O.P. Dahlqvist A. Wieslander Å. J. Biol. Chem. 1994; 269: 23484-23490Abstract Full Text PDF PubMed Google Karlsson O.P. Wieslander A. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). E. coli the were and with with various of and cardiolipin, to in A. laidlawii and S. pneumoniae membranes Wieslander A. A. J. PubMed Google J. J. PubMed Scopus Google Scholar), and and of The in that lipids were activators for the two but to An of and for spMGS only to was to the spMGS but to a very The only on was also for the alMGS at but the activity at above of MGlcDAG was most to a of present in the E. coli cell The total of E. coli lipids present in an was to the of All on the cloned alMGS were in with of the enzyme (2Karlsson O.P. Dahlqvist A. Wieslander Å. J. Biol. Chem. 1994; 269: 23484-23490Abstract Full Text PDF PubMed Google Karlsson O.P. Wieslander A. J. Biol. 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PubMed Scopus Google Scholar) were and of in MurG in A was to for the sequences in with the of spMGS and E. with a of about For a high of residues were in the N-terminal the was by of the sequence a high for the N-terminal and a for the a that was but for a of charge is for MurG with a for the sequence to the an of the alMGS lipid glucosyltransferase to have several structural features in common with certain membrane-binding proteins of known structure, the E. coli The for the well MGlcDAG synthase from A. laidlawii strain A-EF22 was and the encoded catalytic was In the was as the acceptor and as the donor for synthesis of the lipid The of the was characterized by a synthesis of the glucolipid which as S. Karlsson O.P. Wieslander Å. Biochemistry. PubMed Scopus Google Scholar). is present laidlawii PubMed Scopus Google Scholar). Gram-positive the ones in the lipid in membranes and S. Scholar). 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Full Text Full Text PDF PubMed Scopus Google Scholar). from were also for Streptococcus and Streptococcus In the Gram-positive sequences of the MGlcDAG synthase are to gene in an by as glycosyltransferases of CAZy family 4 was to the A. laidlawii gene in the related potential lipid on the and Å. in However, the alMGS sequence was related to the pneumoniae in with the potential group of lipid to widely spread in to the of in and including Gram-positive and and are but the also sequences from eukaryotes. they were also in the and of and archaea, that a common to these was very in the the separation of the bacterial from the All and analyzed contained the EX7E motif typical for the of CAZy family 4 J. J. 1997; PubMed Scopus Google Scholar). A of the in are involved in the synthesis of various lipids enzyme and in in with the of this lipid in PubMed Google Scholar), and the S. pneumoniae the synthase various including in the two ones and S. J. PubMed Scopus Google Scholar). 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In the A. laidlawii this is the In the two clones in the new nonbilayer-prone glucolipid was to The of was to the the of the negatively charged lipids was of the major coli lipid an regulation of the lipid synthesis to certain the spontaneous S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), in the bilayer. However, the nonbilayer-prone from an of only in E. coli A. S. A. 1997; PubMed Scopus Google Scholar). The of in vivo MGlcDAG synthesis in depend on (i) the of in E. coli (ii) a density of amino in to a binding to an anionic (iii) an on the enzyme in in of this enzyme in S. pneumoniae J. S. PubMed Scopus Google Scholar) the glucosyltransferase activity to a that the protein was to the The major nonbilayer-prone lipid in E. as an to this activity to from in been in S. to only a have been The of these are using the mechanism and the in the are in the but one is the determined structure of S. Biol. 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Scholar), with membrane A model for the and of alMGS with a lipid bilayer surface by a of and is in is based on (i) the indicated between the determined MurG glycosyltransferase and alMGS (and on the (ii) the dependence of alMGS activity on the lipid (3Karlsson O.P. Dahlqvist A. Vikstroem S. Wieslander A. J. Biol. Chem. 1997; 272: 929-936Abstract Full Text Full Text PDF PubMed Scopus (34) Google (iii) the corresponding dependence of alMGS binding to (and the to most alMGS from membranes only by and Karlsson and Å. and the of several potential amphipathic segments in the alMGS typical for many lipid proteins and R. Biol. PubMed Scopus Google a close of the to the bilayer the by the and of negatively charged In the enzyme the major nonbilayer-prone membrane lipid MGlcDAG in A. laidlawii is related to a large group of lipid glycosyltransferases in nature. in related bacteria and a structure to E. coli and it is to the membrane by and We Umeå for the S. pneumoniae and we are to and for We are also to Karlsson and for and performed the of the N-terminal and internal sequence of the We for with the sequence were from the for