Identification and analysis of the balhimycin biosynthetic gene cluster and its use for manipulating glycopeptide biosynthesis in Amycolatopsis mediterranei DSM5908. (17/1334)

Seven complete genes and one incomplete gene for the biosynthesis of the glycopeptide antibiotic balhimycin were isolated from the producer, Amycolatopsis mediterranei DSM5908, by a reverse-cloning approach and characterized. Using oligonucleotides derived from glycosyltransferase sequences, a 900-bp glycosyltransferase gene fragment was amplified and used to identify a DNA fragment of 9,882 bp. Of the identified open reading frames, three (oxyA to -C) showed significant sequence similarities to cytochrome P450 monooxygenases and one (bhaA) showed similarities to halogenase, and the genes bgtfA to -C showed similarities to glycosyltransferases. Glycopeptide biosynthetic mutants were created by gene inactivation experiments eliminating oxygenase and glycosyltransferase functions. Inactivation of the oxygenase gene(s) resulted in a balhimycin mutant (SP1-1) which was not able to synthesize an antibiotically active compound. Structural analysis by high-performance liquid chromatography-mass spectrometry, fragmentation studies, and amino acid analysis demonstrated that these oxygenases are involved in the coupling of the aromatic side chains of the unusual heptapeptide. Mutant strain HD1, created by inactivation of the glycosyltransferase gene bgtfB, produced at least four different compounds which were not glycosylated but still antibiotically active.  (+info)

Crystal structures of the bovine beta4galactosyltransferase catalytic domain and its complex with uridine diphosphogalactose. (18/1334)

beta1,4-galactosyltransferase T1 (beta4Gal-T1, EC 2.4.1.90/38), a Golgi resident membrane-bound enzyme, transfers galactose from uridine diphosphogalactose to the terminal beta-N-acetylglucosamine residues forming the poly-N-acetyllactosamine core structures present in glycoproteins and glycosphingolipids. In mammals, beta4Gal-T1 binds to alpha-lactalbumin, a protein that is structurally homologous to lyzozyme, to produce lactose. beta4Gal-T1 is a member of a large family of homologous beta4galactosyltransferases that use different types of glycoproteins and glycolipids as substrates. Here we solved and refined the crystal structures of recombinant bovine beta4Gal-T1 to 2.4 A resolution in the presence and absence of the substrate uridine diphosphogalactose. The crystal structure of the bovine substrate-free beta4Gal-T1 catalytic domain showed a new fold consisting of a single conical domain with a large open pocket at its base. In the substrate-bound complex, the pocket encompassed residues interacting with uridine diphosphogalactose. The structure of the complex contained clear regions of electron density for the uridine diphosphate portion of the substrate, where its beta-phosphate group was stabilized by hydrogen-bonding contacts with conserved residues including the Asp252ValAsp254 motif. These results help the interpretation of engineered beta4Gal-T1 point mutations. They suggest a mechanism possibly involved in galactose transfer and enable identification of the critical amino acids involved in alpha-lactalbumin interactions.  (+info)

Dolichyl sulphate and H-phosphonate: enzymatic reactions with activated sugars. (19/1334)

Two phosphate-modified analogues of dolichyl phosphate were evaluated as substrates or inhibitors of the reactions catalyzed by mammalian microsomal enzymes. Dolichyl H-phosphonate could serve as an efficient acceptor for mannosyl and glucosyl transfer. The reaction products were chromatographically different from those formed from dolichyl phosphate. Lower activity of the H-phosphonate was observed for the reaction of N-acetylglucosaminyl phosphate transfer from UDP-GlcNAc. Dolichyl sulphate was shown not to serve as a substrate for the transfer of mannosyl (from GDP-Man), glucosyl (from UDP-Glc) or N-acetylglucosaminyl phosphate (from UDP-GlcNAc) residues in the presence of rat liver microsomes. Weak inhibitory properties of this analogue were demonstrated.  (+info)

Introducing transglycosylation activity in a liquefying alpha-amylase. (20/1334)

By mutating Ala-289 by Phe or Tyr in the Bacillus stearothermophilus alpha-amylase, we induced this enzyme to perform alcoholytic reactions, a function not present in the wild-type enzyme. This residue was selected from homology analysis with neopullulanase, where the residue has been implicated in the control of transglycosylation [Kuriki et al. (1996) J. Biol. Chem. 271, 17321-173291. We made some inferences about the importance of electrostatic and geometrical modifications in the active site environment of the amylase to explain the behavior of the modified enzyme.  (+info)

Evolutionary considerations in relating oligosaccharide diversity to biological function. (21/1334)

The oligosaccharide chains (glycans) attached to cell surface and extracellular proteins and lipids are known to mediate many important biological roles. However, for many glycans, there are still no evident functions that are of obvious benefit to the organism that synthesizes them. There is also no clear explanation for the extreme complexity and diversity of glycans that can be found on a given glycoconjugate or cell type. Based on the limited information available about the scope and distribution of this diversity among taxonomic groups, it is difficult to see clear trends or patterns consistent with different evolutionary lineages. It appears that closely related species may not necessarily share close similarities in their glycan diversity, and that more derived species may have simpler as well as more complex structures. Intraspecies diversity can also be quite extensive, often without obvious functional relevance. We suggest one general explanation for these observations, that glycan diversification in complex multicellular organisms is driven by evolutionary selection pressures of both endogenous and exogenous origin. We argue that exogenous selection pressures mediated by viral and microbial pathogens and parasites that recognize glycans have played a more prominent role, favoring intra- and interspecies diversity. This also makes it difficult to appreciate and elucidate the specific endogenous roles of the glycans within the organism that synthesizes them.  (+info)

Generation of a novel polysaccharide by inactivation of the aceP gene from the acetan biosynthetic pathway in Acetobacter xylinum. (22/1334)

The acetan biosynthetic pathway in Acetobacter xylinum is an ideal model system for engineering novel bacterial polysaccharides. To genetically manipulate this pathway, an Acetobacter strain (CKE5), more susceptible to gene-transfer methodologies, was developed. A new gene, aceP, involved in acetan biosynthesis was identified, sequenced and shown to have homology at the amino acid level with beta-D-glucosyl transferases from a number of different organisms. Disruption of aceP in strain CKE5 confirmed the function assigned above and was used to engineer a novel polysaccharide with a pentasaccharide repeat unit.  (+info)

Genetic characterization of a Tn5-disrupted glycosyltransferase gene homolog in Brucella abortus and its effect on lipopolysaccharide composition and virulence. (23/1334)

We constructed a rough mutant of Brucella abortus 2308 by transposon (Tn5) mutagenesis. Neither whole cells nor extracted lipopolysaccharide (LPS) from this mutant, designated RA1, reacted with a Brucella O-side-chain-specific monoclonal antibody (MAb), Bru-38, indicating the absence of O-side-chain synthesis. Compositional analyses of LPS from strain RA1 showed reduced levels of quinovosamine and mannose relative to the levels in the parental, wild-type strain, 2308. We isolated DNA flanking the Tn5 insertion in strain RA1 by cloning a 25-kb XbaI genomic fragment into pGEM-3Z to create plasmid pJM6. Allelic exchange of genomic DNA in B. abortus 2308 mediated by electroporation of pJM6 produced kanamycin-resistant clones that were not reactive with MAb Bru-38. Southern blot analysis of genomic DNA from these rough clones revealed Tn5 in a 25-kb XbaI genomic fragment. A homology search with the deduced amino acid sequence of the open reading frame disrupted by Tn5 revealed limited homology with various glycosyltransferases. This B. abortus gene has been named wboA. Transformation of strain RA1 with a broad-host-range plasmid bearing the wild-type B. abortus wboA gene resulted in the restoration of O-side-chain synthesis and the smooth phenotype. B. abortus RA1 was attenuated for survival in mice. However, strain RA1 persisted in mice spleens for a longer time than the B. abortus vaccine strain RB51, but as expected, neither strain induced antibodies specific for the O side chain.  (+info)

Altered mRNA expression of glycosyltransferases in human gastric carcinomas. (24/1334)

Biosynthesis of carbohydrate structures is tissue-specific and developmentally regulated by glycosyltransferases like fucosyl-, sialyl- and N-acetylglucosaminyltransferases. During carcinogenesis, aberrant glycosylation leads to the development of tumor subpopulations with different adhesion properties. The aim of this contribution was to directly compare mRNA expression of several glycosyltransferases in surgical specimens of gastric carcinomas. Carcinoma specimens were classified and characterized according to the WHO/UICC system. In each case, the expression of 12 glycosyltransferase enzymes was studied simultaneously by RT-PCR. For semi-quantitative analysis, amplification of the sample sequence was compared with that of beta-actin, co-amplified within the same tube. Expression of N-acetylglucosaminyltransferase V in gastric carcinomas was significantly enhanced compared to normal tissue. Also, expression of sialyltransferase ST3Gal-IV and fucosyltransferase FT-IV was significantly enhanced in carcinoma tissue. No significant differences in glycosyltransferase expression were found in samples positive for Helicobacter pylori or between the different gastric regions. Thus, carcinogenesis is characterized by specific alterations in mRNA expression of several glycosyltransferases. Future studies will show whether RT-PCR detection of the expression of these enzymes could be helpful for prognostic purposes.  (+info)