Pentaketide resorcylic acid synthesis by type III polyketide synthase from Neurospora crassa

Type III polyketide synthases (PKSs) are responsible for aromatic polyketide synthesis in plants and bacteria. Genome analysis of filamentous fungi has predicted the presence of fungal type III PKSs, although none have thus far been functionally characterized. In the genome of Neurospora crassa, a single open reading frame, NCU04801.1, annotated as a type III PKS was found. In this report, we demonstrate that NCU04801.1 is a novel type III PKS catalyzing the synthesis of pentaketide alkylresorcylic acids. NCU04801.1, hence named 2'-oxoalkylresorcylic acid synthase (ORAS), preferred stearoyl-CoA as a starter substrate and condensed four molecules of malonyl-CoA to give a pentaketide intermediate. For ORAS to yield pentaketide alkylresorcylic acids, aldol condensation and aromatization of the intermediate, which is still attached to the enzyme, are presumably followed by hydrolysis for release of the product as a resorcylic acid. ORAS is the first type III PKS that synthesizes pentaketide resorcylic acids.     

Identification and characterization of multiple curcumin synthases from the herb Curcuma longa

Curcuminoids are pharmaceutically important compounds isolated from the herb Curcuma longa. Two additional type III polyketide synthases, named CURS2 and CURS3, that are capable of curcuminoid synthesis were identified and characterized. In vitro analysis revealed that CURS2 preferred feruloyl-CoA as a starter substrate and CURS3 preferred both feruloyl-CoA and p-coumaroyl-CoA. These results suggested that CURS2 synthesizes curcumin or demethoxycurcumin and CURS3 synthesizes curcumin, bisdemethoxycurcumin and demethoxycurcumin. The availability of the substrates and the expression levels of the three different enzymes capable of curcuminoid synthesis with different substrate specificities might influence the composition of curcuminoids in the turmeric and in different cultivars.     

Gibberellin biosynthesis in bacteria: Separate ent-copalyl diphosphate and ent-kaurene synthases in Bradyrhizobium japonicum

Gibberellins are ent-kaurene-derived diterpenoid phytohormones produced by plants, fungi, and bacteria. The distinct gibberellin biosynthetic pathways in plants and fungi are known, but not that in bacteria. Plants typically use two diterpene synthases to form ent-kaurene, while fungi use only a single bifunctional diterpene synthase. We demonstrate here that Bradyrhizobium japonicum encodes separate ent-copalyl diphosphate and ent-kaurene synthases. These are found in an operon whose enzymatic composition indicates that gibberellin biosynthesis in bacteria represents a third independently assembled pathway relative to plants and fungi. Nevertheless, sequence comparisons also suggest potential homology between diterpene synthases from bacteria, plants, and fungi.     

Alkylresorcinol biosynthesis in plants: New insights from an ancient enzyme family?

Alkylresorcinols are members of an extensive family of bioactive compounds referred to as phenolic lipids, which occur primarily in plants, fungi and bacteria. In plants, alkylresorcinols and their derivatives are thought to serve important roles as phytoanticipins and allelochemicals, although direct evidence for this is still somewhat lacking. Specialized type III polyketide synthases (referred to as ‘alkylresorcinol synthases’), which catalyze the formation of 5-alkylresorcinols using fatty acyl-CoA starter units and malonyl-CoA extender units, have been characterized from several microbial species; however, until very recently little has been known concerning their plant counterparts. Through the use of sorghum and rice EST and genomic data sets, significant inroads have now been made in this regard. Here we provide additional information concerning our recent report on the identification and characterization of alkylresorcinol synthases from Sorghum bicolor and Oryza sativa, as well as a brief consideration of the emergence of this intriguing subfamily of enzymes.Key words: alkylresorcinol, polyketide synthase, alkylresorcinol synthase, phenolic lipid, antifungal     

Biosynthesis of Aliphatic Polyketides by Type III Polyketide Synthase and Methyltransferase in Bacillus subtilis

Type III polyketide synthases (PKSs) synthesize a variety of aromatic polyketides in plants, fungi, and bacteria. The bacterial genome projects predicted that probable type III PKS genes are distributed in a wide variety of gram-positive and -negative bacteria. The gram-positive model microorganism Bacillus subtilis contained the bcsA-ypbQ operon, which appeared to encode a type III PKS and a methyltransferase, respectively. Here, we report the characterization of bcsA (renamed bpsA, for Bacillus pyrone synthase, on the basis of its function) and ypbQ, which are involved in the biosynthesis of aliphatic polyketides. In vivo analysis demonstrated that BpsA was a type III PKS catalyzing the synthesis of triketide pyrones from long-chain fatty acyl-coenzyme A (CoA) thioesters as starter substrates and malonyl-CoA as an extender substrate, and YpbQ was a methyltransferase acting on the triketide pyrones to yield alkylpyrone methyl ethers. YpbQ thus was named BpsB because of its functional relatedness to BpsA. In vitro analysis with histidine-tagged BpsA revealed that it used broad starter substrates and produced not only triketide pyrones but also tetraketide pyrones and alkylresorcinols. Although the aliphatic polyketides were expected to localize in the membrane and play some role in modulating the rigidity and properties of the membrane, no detectable phenotypic changes were observed for a B. subtilis mutant containing a whole deletion of the bpsA-bpsB operon.Type III polyketide synthases (PKSs), represented by a plant chalcone synthase (CHS), are the condensing enzymes that catalyze the synthesis of aromatic polyketides in plants, fungi, and bacteria (2). CHS catalyzes the decarboxylative condensation of p-coumaroyl-coenzyme A (p-coumaroyl-CoA), called a starter substrate, with three malonyl-CoAs, called extender substrates, and synthesizing a tetraketide intermediate. The synthesized tetraketide intermediate was cyclized and aromatized by CHS and resulted in naringenin chalcone. Like CHS, most of the type III PKSs catalyze the condensation of a starter substrate with several molecules of an extender substrate and cyclization. There are many type III PKSs that differ in these specificities.Until recently, type III PKSs were discovered only from plants. In 1999, the first bacterial type III PKS, RppA, was discovered. RppA catalyzes the condensation of five malonyl-CoAs to synthesize 1,3,6,8-tetrahydroxynaphthalene, which is a precursor of hexahydroxyperylenequinone melanin in the actinomycete Streptomyces griseus (4). Since then, the genome projects of various bacteria have revealed that type III PKSs are widely distributed in a variety of bacteria. For example, ArsB and ArsC, both of which are type III PKSs in Azotobacter vinelandii, catalyze the synthesis of alkylresorcinols and alkylpyrones, respectively, which are essential for encystment as the major lipids in the cyst membrane (5). In S. griseus, the srs operon consisting of srsA, srsB, and srsC is responsible for the synthesis of methylated phenolic lipids derived from alkylresorcinols and alkylpyrones (6). The function of each of the operon members is that SrsA is a type III PKS responsible for the synthesis of phenolic lipids alkylresorcinol and alkylpyrones, SrsB is a methyltransferase acting on the phenolic lipids to yield alkylresorcinol methyl ethers, and SrsC is a hydroxylase acting on the alkylresorcinol methyl ethers. The phenolic lipids synthesized by the Srs enzymes confer resistance to β-lactam antibiotics (6). Therefore, it is suggested that phenolic lipids play an important role as minor components in the biological membrane in various bacteria. In fact, srsAB- and srsABC-like operons are distributed widely in both gram-positive and -negative bacteria (see Fig. S1 in the supplemental material). However, most of these type III PKSs have not been characterized.Bacillus subtilis is one of the best-characterized gram-positive bacteria. BcsA, which stands for bacterial chalcone synthase, was annotated as a homologue of type III PKS in B. subtilis (3). As described in this paper, however, this annotation needs correction. We renamed the gene bpsA (for Bacillus pyrone synthase). Moreover, the functional unknown gene ypbQ is located next to bpsA. YpbQ, consisting of 168 amino acid residues, contained an isoprenylcysteine carboxyl methyltransferase (ICMT) domain of the ICMT family members, which are unique membrane proteins that are involved in the posttranslational modification of oncogenic proteins (23). Therefore, the bpsA and ypbQ genes were predicted to form an operon, just like srsA and srsB in the srs operon in S. griseus. We therefore named ypbQ, a thus-far functionally unknown gene, bpsB.In this study, we characterized the functions of BpsA and BpsB by in vivo and in vitro experiments. The in vivo experiments revealed that the overexpression of bpsA in B. subtilis led to the production of triketide pyrones, and the co-overexpression of bpsA and bpsB led to the production of triketide pyrone methyl ethers. The in vitro analysis showed that BpsA produced triketide pyrones and a small amount of tetraketide pyrones and tetraketide resorcinols from long-chain fatty acyl CoA thioesters as starter substrates and malonyl-CoA as an extender substrate. Therefore, BpsA is a type III PKS that is responsible for the synthesis of alkylpyrones, and BpsB is a methyltransferase that acts on the alkylpyrones to yield alkylpyrone methyl ethers. BpsB is the first enzyme found to methylate alkylpyrones. Furthermore, we attempted to analyze the biological function of the aliphatic polyketides by disrupting the bpsA and bpsB genes, but no distinct phenotypic changes were detected under laboratory conditions.     

Structural and functional analysis of C2-type ketoreductases from modular polyketide synthases

The process by which α-stereocenters of polyketide intermediates are set by modular polyketide synthases (PKSs) when condensation is not immediately followed by reduction is mysterious. However, the reductase-incompetent ketoreductase (KR) from the third module of 6-deoxyerythronolide B synthase has been proposed to operate as a racemase, aiding in the epimerization process that reverses the orientation of the α-methyl group of the polyketide intermediate generated by the ketosynthase to the configuration observed in the 6-deoxyerythronolide B final product. To learn more about the epimerization process, the structure of the C2-type KR from the third module of the pikromycin synthase, analogous to the KR from the third module of 6-deoxyerythronolide B synthase, was determined to 1.88 Å resolution. This first structural analysis of this KR-type reveals differences from reductase-competent KRs such as that the site NADPH binds to reductase-competent KRs is occluded by side chains and the putative catalytic tyrosine possesses more degrees of freedom. The active-site geometry may enable C2-type KRs to align the thioester and β-keto groups of a polyketide intermediate to reduce the pK_a of the α-proton and accelerate its abstraction. Results from in vivo assays of engineered PKSs support that C2-type KRs cooperate with epimer-specific ketosynthases to set the configurations of substituent-bearing α-carbons.     

Physcomitrella PpORS,Basal to Plant Type III Polyketide Synthases in Phylogenetic Trees,Is a Very Long Chain 2′-Oxoalkylresorcinol Synthase

The plant type III polyketide synthases (PKSs), which produce diverse secondary metabolites with different biological activities, have successfully co-evolved with land plants. To gain insight into the roles that ancestral type III PKSs played during the early evolution of land plants, we cloned and characterized PpORS from the moss Physcomitrella. PpORS has been proposed to closely resemble the most recent common ancestor of the plant type III PKSs. PpORS condenses a very long chain fatty acyl-CoA with four molecules of malonyl-CoA and catalyzes decarboxylative aldol cyclization to yield the pentaketide 2′-oxoalkylresorcinol. Therefore, PpORS is a 2′-oxoalkylresorcinol synthase. Structure modeling and sequence alignments identified a unique set of amino acid residues (Gln²¹⁸, Val²⁷⁷, and Ala²⁸⁶) at the putative PpORS active site. Substitution of the Ala²⁸⁶ to Phe apparently constricted the active site cavity, and the A286F mutant instead produced triketide alkylpyrones from fatty acyl-CoA substrates with shorter chain lengths. Phylogenetic analysis and comparison of the active sites of PpORS and alkylresorcinol synthases from sorghum and rice suggested that the gramineous enzymes evolved independently from PpORS to have similar functions but with distinct active site architecture. Microarray analysis revealed that PpORS is exclusively expressed in nonprotonemal moss cells. The in planta function of PpORS, therefore, is probably related to a nonprotonemal structure, such as the cuticle.     

Characterization of an alkylresorcinol synthase that forms phenolics accumulating in the cuticular wax on various organs of rye (Secale cereale)

Alkylresorcinols are bioactive compounds produced in diverse plant species, with chemical structures combining an aliphatic hydrocarbon chain and an aromatic ring with characteristic hydroxyl substituents. Here, we aimed to isolate and characterize the enzyme that forms the alkylresorcinols accumulating in the cuticular wax on the surface of all above‐ground organs of rye. Based on sequence homology with other type‐III polyketide synthases, a candidate alkylresorcinol synthase was cloned. Yeast heterologous expression showed that the enzyme, ScARS, is highly specific for the formation of the aromatic resorcinol ring structure, through aldol condensation analogous to stilbene synthases. The enzyme accepts long‐chain and very‐long‐chain acyl‐CoA starter substrates, preferring saturated over unsaturated chains. It typically carries out three rounds of condensation with malonyl‐CoA prior to cyclization, with only very minor activity for a fourth round of malonyl‐CoA condensation and cyclization to 5‐(2′‐oxo)‐alkylresorcinols or 5‐(2′‐hydroxy)‐alkylresorcinols. Like other enzymes involved in cuticle formation, ScARS is localized to the endoplasmic reticulum. ScARS expression patterns were found correlated with alkylresorcinol accumulation during leaf development and across different rye organs. Overall, our results thus suggest that ScARS synthesizes the cuticular alkylresorcinols found on diverse rye organ surfaces.     

Production of fully assembled and active Aquifex aeolicus F1FO ATP synthase in Escherichia coli

Background

F₁F_O ATP synthases catalyze the synthesis of ATP from ADP and inorganic phosphate driven by ion motive forces across the membrane. A number of ATP synthases have been characterized to date. The one from the hyperthermophilic bacterium Aquifex aeolicus presents unique features, i.e. a putative heterodimeric stalk. To complement previous work on the native form of this enzyme, we produced it heterologously in Escherichia coli.

Methods

We designed an artificial operon combining the nine genes of A. aeolicus ATP synthase, which are split into four clusters in the A. aeolicus genome. We expressed the genes and purified the enzyme complex by affinity and size-exclusion chromatography. We characterized the complex by native gel electrophoresis, Western blot, and mass spectrometry. We studied its activity by enzymatic assays and we visualized its structure by single-particle electron microscopy.

Results

We show that the heterologously produced complex has the same enzymatic activity and the same structure as the native ATP synthase complex extracted from A. aeolicus cells. We used our expression system to confirm that A. aeolicus ATP synthase possesses a heterodimeric peripheral stalk unique among non-photosynthetic bacterial F₁F_O ATP synthases.

Conclusions

Our system now allows performing previously impossible structural and functional studies on A. aeolicus F₁F_O ATP synthase.

General significance

More broadly, our work provides a valuable platform to characterize many other membrane protein complexes with complicated stoichiometry, i.e. other respiratory complexes, the nuclear pore complex, or transporter systems.     

Aspergillus oryzae type III polyketide synthase CsyB uses a fatty acyl starter for the biosynthesis of csypyrone B compounds

Csypyrones B1, B2 and B3 are α-pyrones that can be obtained from Aspergillus oryzae expressing CsyB, which is a type III polyketide synthase. We investigated the biosynthesis of the csypyrone B compounds using [1-¹³C] and [2-¹³C] acetate feeding experiments. ¹³C NMR analyses of the methyl esters of the csypyrone B compounds fed with the ¹³C-labeled acetates showed that the carboxyl carbons of the csypyrone B side-chains were derived from the C-2 methyl carbon of the acetate. These results indicated that fatty acyl starters are involved in the CsyB reaction and that the csypyrone B compounds are formed by the oxidation of side-chains by the host fungus.     

Enzymatic formation of an aromatic dodecaketide by engineered plant polyketide synthase

Octaketide synthase (OKS) from Aloe arborescens is a plant-specific type III polyketide synthase (PKS) that catalyzes iterative condensations of eight molecules of malonyl-CoA to produce the C₁₆ aromatic octaketides SEK4 and SEK4b. On the basis of the crystal structures of OKS, the F66L/N222G double mutant was constructed and shown to produce an unnatural dodecaketide TW95a by sequential condensations of 12 molecules of malonyl-CoA. The C₂₄ naphthophenone TW95a is a product of the minimal type II PKS (whiE from Streptomyces coelicolor), and is structurally related to the C₂₀ decaketide benzophenone SEK15, the product of the OKS N222G point mutant. The C₂₄ dodecaketide naphthophenone TW95a is the first and the longest polyketide scaffold generated by a structurally simple type III PKS. A homology model predicted that the active-site cavity volume of the F66L/N222G mutant is increased to 748 Å³, from 652 Å³ of the wild-type OKS. The structure-based engineering thus greatly expanded the catalytic repertoire of the simple type III PKS to further produce larger and more complex polyketide molecules.     

Aspergillus oryzae type III polyketide synthase CsyA is involved in the biosynthesis of 3,5-dihydroxybenzoic acid

As a novel superfamily of type III polyketide synthases in microbes, four genes csyA, csyB, csyC, and csyD, were found in the genome of Aspergillus oryzae, an industrially important filamentous fungus. In order to analyze their functions, we carried out the overexpression of csyA under the control of α-amylase promoter in A. oryzae and identified 3,5-dihydroxybenzoic acid (DHBA) as the major product. Feeding experiments using ¹³C-labeled acetates confirmed that the acetate labeling pattern of DHBA coincided with that of orcinol derived from orsellinic acid, a polyketide formed by the condensation and cyclization of four acetate units. Further oxidation of methyl group of orcinol by the host fungus could lead to the production of DHBA. Comparative molecular modeling of CsyA with the crystal structure of Neurospora crassa 2′-oxoalkylresorcylic acid synthase indicated that CsyA cavity size can only accept short-chain acyl starter and tetraketide formation. Thus, CsyA is considered to be a tetraketide alkyl-resorcinol/resorcylic acid synthase.     

The O-methyltransferase SrsB catalyzes the decarboxylative methylation of alkylresorcylic acid during phenolic lipid biosynthesis by Streptomyces griseus

Streptomyces griseus contains the srs operon, which is required for phenolic lipid biosynthesis. The operon consists of srsA, srsB, and srsC, which encode a type III polyketide synthase, an O-methyltransferase, and a flavoprotein hydroxylase, respectively. We previously reported that the recombinant SrsA protein synthesized 3-(13'-methyltetradecyl)-4-methylresorcinol, using iso-C(16) fatty acyl-coenzyme A (CoA) as a starter substrate and malonyl-CoA and methylmalonyl-CoA as extender substrates. An in vitro SrsA reaction using [(13)C(3)]malonyl-CoA confirmed that the order of extender substrate condensation was methylmalonyl-CoA, followed by two extensions with malonyl-CoA. Furthermore, SrsA was revealed to produce an alkylresorcylic acid as its direct product rather than an alkylresorcinol. The functional SrsB protein was produced in the membrane fraction in Streptomyces lividans and used for the in vitro SrsB reaction. When the SrsA reaction was coupled, SrsB produced alkylresorcinol methyl ether in the presence of S-adenosyl-l-methionine (SAM). SrsB was incapable of catalyzing the O-methylation of alkylresorcinol, indicating that alkylresorcylic acid was the substrate of SrsB and that SrsB catalyzed the conversion of alkylresorcylic acid to alkylresorcinol methyl ether, namely, by both the O-methylation of the hydroxyl group (C-6) and the decarboxylation of the neighboring carboxyl group (C-1). O-methylated alkylresorcylic acid was not detected in the in vitro SrsAB reaction, although it was presumably stable, indicating that O-methylation did not precede decarboxylation. We therefore postulated that O-methylation was coupled with decarboxylation and proposed that SrsB catalyzed the feasible SAM-dependent decarboxylative methylation of alkylresorcylic acid. To the best of our knowledge, this is the first report of a methyltransferase that catalyzes decarboxylative methylation.     

Cloning and characterization of a type III polyketide synthase from Aspergillus niger

Type III polyketide synthases (PKSs) are the condensing enzymes that catalyze the formation of a myriad of aromatic polyketides in plant, bacteria, and fungi. Here we report the cloning and characterization of a putative type III PKS from Aspergillusniger, AnPKS. This enzyme catalyzes the synthesis of alkyl pyrones from C2 to C18 starter CoA thioesters with malonyl-CoA as an extender CoA through decaboxylative condensation and cyclization. It displays broad substrate specificity toward fatty acyl-CoA starters to yield triketide and tetraketide pyrones, with benzoyl-CoA as the most preferred starter. The optimal temperature and pH of AnPKS are 50°C and 8, respectively. Under optimal conditions, the enzyme shows the highest catalytic efficiency (k(cat)/K(m)) of 7.4×10(5)s(-1)M(-1) toward benzoyl-CoA. Homology modeling and site-directed mutagenesis were used to probe the molecular basis of its substrate specificity. This study should open doors for further engineering of AnPKS as a biocatalyst for synthesis of value-added polyketides.     

Discovery of a novel superfamily of type III polyketide synthases in Aspergillus oryzae

Identification of genes encoding type III polyketide synthase (PKS) superfamily members in the industrially useful filamentous fungus, Aspergillus oryzae, revealed that their distribution is not specific to plants or bacteria. Among other Aspergilli (Aspergillus nidulans and Aspergillus fumigatus), A. oryzae was unique in possessing four chalcone synthase (CHS)-like genes (csyA, csyB, csyC, and csyD). Expression of csyA, csyB, and csyD genes was confirmed by RT-PCR. Comparative genome analyses revealed single putative type III PKS in Neurospora crassa and Fusarium graminearum, two each in Magnaporthe grisea and Podospora anserina, and three in Phenarocheate chrysosporium, with a phylogenic distinction from bacteria and plants. Conservation of catalytic residues in the CHSs across species implicated enzymatically active nature of these newly discovered homologs.     

Aspergillus oryzae CsyB Catalyzes the Condensation of Two β-Ketoacyl-CoAs to Form 3-Acetyl-4-hydroxy-6-alkyl-α-pyrone

The type III polyketide synthases from fungi produce a variety of secondary metabolites including pyrones, resorcinols, and resorcylic acids. We previously reported that CsyB from Aspergillus oryzae forms α-pyrone csypyrone B compounds when expressed in A. oryzae. Feeding experiments of labeled acetates indicated that a fatty acyl starter is involved in the reaction catalyzed by CsyB. Here we report the in vivo and in vitro reconstitution analysis of CsyB. When CsyB was expressed in Escherichia coli, we observed the production of 3-acetyl-4-hydroxy-α-pyrones with saturated or unsaturated straight aliphatic chains of C₉–C₁₇ in length at the 6 position. Subsequent in vitro analysis using recombinant CsyB revealed that CsyB could accept butyryl-CoA as a starter substrate and malonyl-CoA and acetoacetyl-CoA as extender substrates to form 3-acetyl-4-hydroxy-6-propyl-α-pyrone. CsyB also afforded dehydroacetic acid from two molecules of acetoacetyl-CoA. Furthermore, synthetic N-acetylcysteamine thioester of β-ketohexanoic acid was converted to 3-butanoyl-4-hydroxy-6-propyl-α-pyrone by CsyB. These results therefore confirmed that CsyB catalyzed the synthesis of β-ketoacyl-CoA from the reaction of the starter fatty acyl CoA thioesters with malonyl-CoA as the extender through decarboxylative condensation and further coupling with acetoacetyl-CoA to form 3-acetyl-4-hydroxy-6-alkyl-α-pyrone. CsyB is the first type III polyketide synthase that synthesizes 3-acetyl-4-hydroxy-6-alkyl-α-pyrone by catalyzed the coupling of two β-ketoacyl-CoAs.     

Characterisation of Desulfovibrio vulgaris haem b synthase,a radical SAM family member

An alternative route for haem b biosynthesis is operative in sulfate-reducing bacteria of the Desulfovibrio genus and in methanogenic Archaea. This pathway diverges from the canonical one at the level of uroporphyrinogen III and progresses via a distinct branch, where sirohaem acts as an intermediate precursor being converted into haem b by a set of novel enzymes, named the alternative haem biosynthetic proteins (Ahb). In this work, we report the biochemical characterisation of the Desulfovibrio vulgaris AhbD enzyme that catalyses the last step of the pathway. Mass spectrometry analysis showed that AhbD promotes the cleavage of S-adenosylmethionine (SAM) and converts iron-coproporphyrin III via two oxidative decarboxylations to yield haem b, methionine and the 5′-deoxyadenosyl radical. Electron paramagnetic resonance spectroscopy studies demonstrated that AhbD contains two [4Fe–4S]^2 +/1 + centres and that binding of the substrates S-adenosylmethionine and iron-coproporphyrin III induces conformational modifications in both centres. Amino acid sequence comparisons indicated that D. vulgaris AhbD belongs to the radical SAM protein superfamily, with a GGE-like motif and two cysteine-rich sequences typical for ligation of SAM molecules and iron-sulfur clusters, respectively. A structural model of D. vulgaris AhbD with putative binding pockets for the iron-sulfur centres and the substrates SAM and iron-coproporphyrin III is discussed.     

Efficient transfer of sialo-oligosaccharide onto proteins by combined use of a glycosynthase-like mutant of Mucor hiemalis endoglycosidase and synthetic sialo-complex-type sugar oxazoline

Background

An efficient method for synthesizing homogenous glycoproteins is essential for elucidating the structural and functional roles of glycans of glycoproteins. We have focused on the transglycosylation activity of endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) as a tool for glycoconjugate syntheses, since it can transfer en bloc the oligosaccharide of not only high-mannose type but also complex-type N-glycan onto various acceptors having an N-acetylglucosamine residue. However, there are two major bottlenecks for its practical application: the low yield of the transglycosylation product and the difficulty to obtain the activated sugar oxazoline substrate, especially the sialo-complex type one.

Methods

We carried out the transglycosylation using a glycosynthase-like N175Q mutant of Endo-M, which was found to possess enhanced transglycosylation activity with sugar oxazoline as a donor substrate, in combination with an easy preparation of the sialo-complex-type sugar oxazoline from natural sialoglycopeptide in egg yolk.

Results

Endo-M-N175Q showed efficient transglycosylation toward sialo-complex-type sugar oxazoline onto bioactive peptides and bovine ribonuclease B, and each sialylated compound was obtained in significantly high yield.

Conclusions

Highly efficient and simple chemo-enzymatic syntheses of various sialylated compounds were enabled, by a combination of a simple synthesis of sialo-complex-type sugar oxazoline and the Endo-M-N175Q catalyzed transglycosylation.

General significance

Our method would be very useful for a practical synthesis of biologically important glycopeptides and glycoproteins.