Genetic disruption of multiple α1,2-mannosidases generates mammalian cells producing recombinant proteins with high-mannose-type N-glycans.

Recombinant therapeutic proteins are becoming very important pharmaceutical agents for treating intractable diseases. Most biopharmaceutical proteins are produced in mammalian cells because this ensures correct folding and glycosylation for protein stability and function. However, protein production in mammalian cells has several drawbacks, including heterogeneity of glycans attached to the produced protein. In this study, we established cell lines with high-mannose-type N-linked, low-complexity glycans. We first knocked out two genes encoding Golgi mannosidases (MAN1A1 and MAN1A2) in HEK293 cells. Single knockout (KO) cells did not exhibit changes in N-glycan structures, whereas double KO cells displayed increased high-mannose-type and decreased complex-type glycans. In our effort to eliminate the remaining complex-type glycans, we found that knocking out a gene encoding the endoplasmic reticulum mannosidase I (MAN1B1) in the double KO cells reduced most of the complex-type glycans. In triple KO (MAN1A1, MAN1A2, and MAN1B1) cells, Man9GlcNAc2 and Man8GlcNAc2 were the major N-glycan structures. Therefore, we expressed two lysosomal enzymes, α-galactosidase-A and lysosomal acid lipase, in the triple KO cells and found that the glycans on these enzymes were sensitive to endoglycosidase H treatment. The N-glycan structures on recombinant proteins expressed in triple KO cells were simplified and changed from complex types to high-mannose types at the protein level. Our results indicate that the triple KO HEK293 cells are suitable for producing recombinant proteins, including lysosomal enzymes with high-mannose-type N-glycans.

Alternative routes for synthesis of N-linked glycans by Alg2 mannosyltransferase.

Asparagine ( N)-linked glycosylation requires the ordered, stepwise synthesis of lipid-linked oligosaccharide (LLO) precursor Glc3Man9GlcNAc2-pyrophosphate-dolichol (Glc3Man9Gn2-PDol) on the endoplasmic reticulum. The fourth and fifth steps of LLO synthesis are catalyzed by Alg2, an unusual mannosyltransferase (MTase) with two different MTase activities; Alg2 adds both an α1,3- and α1,6-mannose onto ManGlcNAc2-PDol to form the trimannosyl core Man3GlcNAc2-PDol. The biochemical properties of Alg2 are controversial and remain undefined. In this study, a liquid chromatography/mass spectrometry-based quantitative assay was established and used to analyze the MTase activities of purified yeast Alg2. Alg2-dependent Man3GlcNAc2-PDol production relied on net-neutral lipids with a propensity to form bilayers. We further showed addition of the α1,3- and α1,6-mannose can occur independently in either order but at differing rates. The conserved C-terminal EX7E motif, N-terminal cytosolic tail, and 3 G-rich loop motifs in Alg2 play crucial roles for these activities, both in vitro and in vivo. These findings provide insight into the unique bifunctionality of Alg2 during LLO synthesis and lead to a new model in which alternative, independent routes exist for Alg2 catalysis of the trimannosyl core oligosaccharide.-Li, S.-T., Wang, N., Xu, X.-X., Fujita, M., Nakanishi, H., Kitajima, T., Dean, N., Gao, X.-D. Alternative routes for synthesis of N-linked glycans by Alg2 mannosyltransferase.

Structural and functional analysis of Alg1 beta-1,4 mannosyltransferase reveals the physiological importance of its membrane topology.

In eukaryotes, the biosynthesis of a highly conserved dolichol-linked oligosaccharide (DLO) precursor Glc3Man9GlcNAc2-pyrophosphate-dolichol (PP-Dol) begins on the cytoplasmic face of the endoplasmic reticulum (ER) and ends within the lumen. Two functionally distinguished heteromeric glycosyltransferase (GTase) complexes are responsible for the cytosolic DLO assembly. Alg1, a ß-1, 4 mannosyltransferase (MTase) physically interacts with Alg2 and Alg11 proteins to form the multienzyme complex which catalyzes the addition of all five mannose to generate the Man5GlcNAc2-PP-Dol intermediate. Despite the fact that Alg1 plays a central role in the formation of the multi-MTase has been confirmed, the topological information of Alg1 including the molecular mechanism of membrane association are still poorly understood. Using a combination of bioinformatics and biological approaches, we have undertaken a structural and functional study on Alg1 protein, in which the enzymatic activities of Alg1 and its variants were monitored by a complementation assay using the GALpr-ALG1 yeast strain, and further confirmed by a liquid chromatography-mass spectrometry-based in vitro quantitative assay. Computational and experimental evidence confirmed Alg1 shares structure similarity with Alg13/14 complex, which has been defined as a membrane-associated GT-B GTase. Particularly, we provide clear evidence that the N-terminal transmembrane domain including the following positively charged amino acids and an N-terminal amphiphilic-like α helix domain exposed on the protein surface strictly coordinate the Alg1 orientation on the ER membrane. This work provides detailed membrane topology of Alg1 and further reveals its biological importance at the spatial aspect in coordination of cytosolic DLO biosynthesis.

Effects of Rho1, a small GTPase on the production of recombinant glycoproteins in Saccharomyces cerevisiae.

BACKGROUND: To humanize yeast N-glycosylation pathways, genes involved in yeast specific hyper-mannosylation must be disrupted followed by the introduction of genes catalyzing the synthesis, transport, and addition of human sugars. However, deletion of these genes, for instance, OCH1, which initiates hyper-mannosylation, could cause severe defects in cell growth, morphogenesis and response to environmental challenges. RESULTS: In this study, overexpression of RHO1, which encodes the Rho1p small GTPase, is confirmed to partially recover the growth defect of Saccharomyces cerevisiae Δalg3Δoch1 double mutant strain. In addition, transmission electron micrographs indicated that the cell wall structure of RHO1-expressed cells have an enhanced glucan layer and also a recovered mannoprotein layer, revealing the effect of Rho1p GTPase on cell wall biosynthesis. Similar complementation phenotypes have been confirmed by overexpression of the gene that encodes Fks2 protein, a catalytic subunit of a 1,3-ß-glucan synthase. Besides the recovery of cell wall structure, the RHO1-overexpressed Δalg3Δoch1 strain also showed improved abilities in temperature tolerance, osmotic potential and drug sensitivity, which were not observed in the Δalg3Δoch1-FKS2 cells. Moreover, RHO1 overexpression could also increase N-glycan site occupancy and the amount of secreted glycoproteins. CONCLUSIONS: Overexpression of RHO1 in 'humanized' glycoprotein producing yeasts could significantly facilitate its future industrial applications for the production of therapeutic glycoproteins.

Cytotoxic mechanism of selenomethionine in yeast.

Although selenium is an essential element, its excessive uptake is detrimental to living organisms. The significance of selenium for living organisms has been exploited for various purposes. However, the molecular basis of selenium toxicity is not completely understood. Here, we applied a capillary electrophoresis time-of-flight mass spectrometry-based metabolomics approach to analysis of yeast cells treated with selenomethionine. The data indicated that intracellular thiol compounds are significantly decreased, and diselenide and selenosulfide compounds are increased in selenomethionine-treated cells. The growth defect induced by selenomethionine was recovered by extracellular addition of cysteine and by genetic modification of yeast cells that have an additional de novo synthetic pathway for cysteine. Because cysteine is an intermediate of thiol compounds, these results suggested that the loss of a reduced form of thiol compounds due to selenomethionine causes a growth defect of yeast cells.

Free oligosaccharides to monitor glycoprotein endoplasmic reticulum-associated degradation in Saccharomyces cerevisiae.

In eukaryotic cells, N-glycosylation has been recognized as one of the most common and functionally important co- or post-translational modifications of proteins. "Free" forms of N-glycans accumulate in the cytosol of mammalian cells, but the precise mechanism for their formation and degradation remains unknown. Here, we report a method for the isolation of yeast free oligosaccharides (fOSs) using endo-beta-1,6-glucanase digestion. fOSs were undetectable in cells lacking PNG1, coding the cytoplasmic peptide:N-glycanase gene, suggesting that almost all fOSs were formed from misfolded glycoproteins by Png1p. Structural studies revealed that the most abundant fOS was M8B, which is not recognized well by the endoplasmic reticulum-associated degradation (ERAD)-related lectin, Yos9p. In addition, we provide evidence that some of the ERAD substrates reached the Golgi apparatus prior to retrotranslocation to the cytosol. N-Glycan structures on misfolded glycoproteins in cells lacking the cytosol/vacuole alpha-mannosidase, Ams1p, was still quite diverse, indicating that processing of N-glycans on misfolded glycoproteins was more complex than currently envisaged. Under ER stress, an increase in fOSs was observed, whereas levels of M7C, a key glycan structure recognized by Yos9p, were unchanged. Our method can thus provide valuable information on the molecular mechanism of glycoprotein ERAD in Saccharomyces cerevisiae.

Mutation of high-affinity methionine permease contributes to selenomethionyl protein production in Saccharomyces cerevisiae.

The production of selenomethionine (SeMet) derivatives of recombinant proteins allows phase determination by single-wavelength or multiwavelength anomalous dispersion phasing in X-ray crystallography, and this popular approach has permitted the crystal structures of numerous proteins to be determined. Although yeast is an ideal host for the production of large amounts of eukaryotic proteins that require posttranslational modification, the toxic effects of SeMet often interfere with the preparation of protein derivatives containing this compound. We previously isolated a mutant strain (SMR-94) of the methylotrophic yeast Pichia pastoris that is resistant to both SeMet and selenate and demonstrated its applicability for the production of proteins suitable for X-ray crystallographic analysis. However, the molecular basis for resistance to SeMet by the SMR-94 strain remains unclear. Here, we report the characterization of SeMet-resistant mutants of Saccharomyces cerevisiae and the identification of a mutant allele of the MUP1 gene encoding high-affinity methionine permease, which confers SeMet resistance. Although the total methionine uptake by the mup1 mutant (the SRY5-7 strain) decreased to 47% of the wild-type level, it was able to incorporate SeMet into the overexpressed epidermal growth factor peptide with 73% occupancy, indicating the importance of the moderate uptake of SeMet by amino acid permeases other than Mup1p for the alleviation of SeMet toxicity. In addition, under standard culture conditions, the mup1 mutant showed higher productivity of the SeMet derivative relative to other SeMet-resistant mutants. Based on these results, we conclude that the mup1 mutant would be useful for the preparation of selenomethionyl proteins for X-ray crystallography.

Use of novel selenomethionine-resistant yeast to produce selenomethionyl protein suitable for structural analysis.

Yeast is widely used to determine the tertiary structure of eukaryotic proteins, because of its ability to undergo post-translational modifications such as glycosylation. A mutant lacking S-adenosylmethionine synthesis has been reported as a suitable host for producing selenomethionine derivatives, which can help solve phase problems in protein crystallography. However, the mutant required external addition of S-adenosylmethionine for cell proliferation. Here, a selenomethionine-resistant Pichia pastoris mutant that showed S-adenosylmethionine autotrophy was isolated. Human lysozyme expressed by the mutant under the control of constitutive promoter contained selenomethionine at 65% occupancy, sufficient for use as a selenomethionine derivative for single-wavelength anomalous dispersion phasing.

Glycoengineering of HEK293 cells to produce high-mannose-type N-glycan structures.

Therapeutic proteins are a developing part of the modern biopharmaceutical industry, providing novel therapies to intractable diseases including cancers and autoimmune diseases. The human embryonic kidney 293 (HEK293) cell line has been widely used to produce recombinant proteins in both basic science and industry. The heterogeneity of glycan structures is one of the most challenging issues in the production of therapeutic proteins. Previously, we knocked out genes encoding α1,2-mannosidase-Is, MAN1A1, MAN1A2 and MAN1B1, in HEK293 cells, establishing a triple-knockout (T-KO) cell line, which produced recombinant protein with mainly high-mannose-type N-glycans. Here, we further knocked out MAN1C1 and MGAT1 encoding another Golgi α1,2-mannosidase-I and N-acetylglucosaminyltransferase-I, respectively, based on the T-KO cells. Two recombinant proteins, lysosomal acid lipase (LIPA) and immunoglobulin G1 (IgG1), were expressed in the quadruple-KO (QD-KO) and quintuple-KO (QT-KO) cell lines. Glycan structural analysis revealed that all the hybrid-type and complex-type N-glycans were eliminated, and only the high-mannose-type N-glycans were detected among the recombinant proteins prepared from the QD-KO and QT-KO cells. Overexpression of the oncogenes MYC and MYCN recovered the slow growth in QD-KO and QT-KO without changing the glycan structures. Our results suggest that these cell lines could be suitable platforms to produce homogeneous therapeutic proteins.

Reconstitution of the lipid-linked oligosaccharide pathway for assembly of high-mannose N-glycans.

The asparagine (N)-linked Man9GlcNAc2 is required for glycoprotein folding and secretion. Understanding how its structure contributes to these functions has been stymied by our inability to produce this glycan as a homogenous structure of sufficient quantities for study. Here, we report the high yield chemoenzymatic synthesis of Man9GlcNAc2 and its biosynthetic intermediates by reconstituting the eukaryotic lipid-linked oligosaccharide (LLO) pathway. Endoplasmic reticulum mannosyltransferases (MTases) are expressed in E. coli and used for mannosylation of the dolichol mimic, phytanyl pyrophosphate GlcNAc2. These recombinant MTases recognize unique substrates and when combined, synthesize end products that precisely mimic those in vivo, demonstrating that ordered assembly of LLO is due to the strict enzyme substrate specificity. Indeed, non-physiological glycans are produced only when the luminal MTases are challenged with cytosolic substrates. Reconstitution of the LLO pathway to synthesize Man9GlcNAc2 in vitro provides an important tool for functional studies of the N-linked glycoprotein biosynthesis pathway.

Construction of green fluorescence protein mutant to monitor STT3B-dependent N-glycosylation.

Oligosaccharyltransferases (OSTs) mediate the en bloc transfer of N-glycan intermediates onto the asparagine residue in glycosylation sequons (N-X-S/T, X≠P). These enzymes are typically heteromeric complexes composed of several membrane-associated subunits, in which STT3 is highly conserved as a catalytic core. Metazoan organisms encode two STT3 genes (STT3A and STT3B) in their genome, resulting in the formation of at least two distinct OST isoforms consisting of shared subunits and complex specific subunits. The STT3A isoform of OST primarily glycosylates substrate polypeptides cotranslationally, whereas the STT3B isoform is involved in cotranslational and post-translocational glycosylation of sequons that are skipped by the STT3A isoform. Here, we describe mutant constructs of monomeric enhanced green fluorescent protein (mEGFP), which are susceptible to STT3B-dependent N-glycosylation. The endoplasmic reticulum-localized mEGFP (ER-mEGFP) mutants contained an N-glycosylation sequon at their C-terminus and exhibited increased fluorescence in response to N-glycosylation. Isoform-specific glycosylation of the constructs was confirmed by using STT3A- or STT3B-knockout cell lines. Among the mutant constructs that we tested, the ER-mEGFP mutant containing the N185 -C186 -T187 sequon was the best substrate for the STT3B isoform in terms of glycosylation efficiency and fluorescence change. Our results suggest that the mutant ER-mEGFP is useful for monitoring STT3B-dependent post-translocational N-glycosylation in cells of interest, such as those from putative patients with a congenital disorder of glycosylation.

The genomes of Crithidia bombi and C. expoeki, common parasites of bumblebees.

Trypanosomatids (Trypanosomatidae, Kinetoplastida) are flagellated protozoa containing many parasites of medical or agricultural importance. Among those, Crithidia bombi and C. expoeki, are common parasites in bumble bees around the world, and phylogenetically close to Leishmania and Leptomonas. They have a simple and direct life cycle with one host, and partially castrate the founding queens greatly reducing their fitness. Here, we report the nuclear genome sequences of one clone of each species, extracted from a field-collected infection. Using a combination of Roche 454 FLX Titanium, Pacific Biosciences PacBio RS, and Illumina GA2 instruments for C. bombi, and PacBio for C. expoeki, we could produce high-quality and well resolved sequences. We find that these genomes are around 32 and 34 MB, with 7,808 and 7,851 annotated genes for C. bombi and C. expoeki, respectively-which is somewhat less than reported from other trypanosomatids, with few introns, and organized in polycistronic units. A large fraction of genes received plausible functional support in comparison primarily with Leishmania and Trypanosoma. Comparing the annotated genes of the two species with those of six other trypanosomatids (C. fasciculata, L. pyrrhocoris, L. seymouri, B. ayalai, L. major, and T. brucei) shows similar gene repertoires and many orthologs. Similar to other trypanosomatids, we also find signs of concerted evolution in genes putatively involved in the interaction with the host, a high degree of synteny between C. bombi and C. expoeki, and considerable overlap with several other species in the set. A total of 86 orthologous gene groups show signatures of positive selection in the branch leading to the two Crithidia under study, mostly of unknown function. As an example, we examined the initiating glycosylation pathway of surface components in C. bombi, finding it deviates from most other eukaryotes and also from other kinetoplastids, which may indicate rapid evolution in the extracellular matrix that is involved in interactions with the host. Bumble bees are important pollinators and Crithidia-infections are suspected to cause substantial selection pressure on their host populations. These newly sequenced genomes provide tools that should help better understand host-parasite interactions in these pollinator pathogens.

Structural modeling and mutagenesis of endo-ß-N-acetylglucosaminidase from Ogataea minuta identifies the importance of Trp295 for hydrolytic activity.

Endo-ß-N-acetylglucosaminidase from the methylotrophic yeast Ogataea minuta (Endo-Om) is a glycoside hydrolase family 85 enzyme that has dual catalytic activity in the hydrolysis and transglycosylation of complex N-glycans, in common with the enzymes from the eukaryotic species. In this study, we have conducted mutagenesis of Endo-Om at Trp295, to determine the effect on hydrolytic activity. Structural modeling predicted that Trp295 forms an important interaction with the α-1,3-linked mannose residue of the trimannosyl N-glycan core, rather than being directly involved in catalytic activity. Our results showed that an aromatic amino acid is required at position 295 for the hydrolytic activity of this enzyme. Notably, the tryptophan residue is highly conserved in eukaryotic endo-ß-N-acetylglucosaminidases that show activity toward complex oligosaccharides. Accordingly, our results strongly suggested that Trp295 is involved in the recognition of oligosaccharide substrates by Endo-Om.

N-Glycan-dependent protein folding and endoplasmic reticulum retention regulate GPI-anchor processing.

Glycosylphosphatidylinositol (GPI) anchoring of proteins is a conserved posttranslational modification in the endoplasmic reticulum (ER). Soon after GPI is attached, an acyl chain on the GPI inositol is removed by post-GPI attachment to proteins 1 (PGAP1), a GPI-inositol deacylase. This is crucial for switching GPI-anchored proteins (GPI-APs) from protein folding to transport states. We performed haploid genetic screens to identify factors regulating GPI-inositol deacylation, identifying seven genes. In particular, calnexin cycle impairment caused inefficient GPI-inositol deacylation. Calnexin was specifically associated with GPI-APs, dependent on N-glycan and GPI moieties, and assisted efficient GPI-inositol deacylation by PGAP1. Under chronic ER stress caused by misfolded GPI-APs, inositol-acylated GPI-APs were exposed on the cell surface. These results indicated that N-glycans participate in quality control and temporal ER retention of GPI-APs, ensuring their correct folding and GPI processing before exiting from the ER. Once the system is disrupted by ER stress, unprocessed GPI-APs become exposed on the cell surface.

Saccharomyces cerevisiae alpha1,6-mannosyltransferase has a catalytic potential to transfer a second mannose molecule.

In yeast, the N-linked oligosaccharide modification in the Golgi apparatus is initiated by alpha1,6-mannosyltransferase (encoded by the OCH1 gene) with the addition of mannose to the Man(8)GlcNAc(2) or Man(9)GlcNAc(2) endoplasmic reticulum intermediates. In order to characterize its enzymatic properties, the soluble form of the recombinant Och1p was expressed in the methylotrophic yeast Pichia pastoris as a secreted protein, after truncation of its transmembrane region and fusion with myc and histidine tags at the C-terminus, and purified using a metal chelating column. The enzymatic reaction was performed using various kinds of pyridylaminated (PA) sugar chains as acceptor, and the products were separated by high performance liquid chromatography. The recombinant Och1p efficiently transferred a mannose to Man(8)GlcNAc(2)-PA and Man(9)GlcNAc(2)-PA acceptors, while Man(5)GlcNAc(2)-PA, which completely lacks alpha1,2-linked mannose residues, was not used as an acceptor. At high enzyme concentrations, a novel product was detected by HPLC. Analysis of the product revealed that a second mannose was attached at the 6-O-position of alpha1,3-linked mannose branching from the alpha1,6-linked mannose that is attached to beta1,4-linked mannose of Man(10)GlcNAc(2)-PA produced by the original activity of Och1p. Our results indicate that Och1p has the potential to transfer two mannoses from GDP-mannose, and strictly recognizes the overall structure of high mannose type oligosaccharide.

Selenomethionine metabolism and its toxicity in yeast.

The importance of selenium for organisms can be explained by its existence as selenocysteine in the catalytic centers of glutathione peroxidase and thioredoxin reductase. Another selenoamino acid, selenomethionine, is the major form of selenium in foods, and organisms that require selenium as a nutrient directly metabolize selenomethionine to a reactive form of selenium or store it in general proteins. Selenium is recognized as an essential nutrient for human and animal health; however, its excessive uptake harms mammals and the cytotoxic mechanism of selenium remains unclear. Recent progress in the development of selenium-enriched yeast and selenomethionine-resistant mutant to produce selenomethionine-containing proteins for X-ray crystallography has provided new insights into the molecular mechanism of selenomethionine toxicity. In this review, we describe the metabolism of seleno-compounds in yeast and discuss the cytotoxicity caused by selenomethionine against yeast from a metabolic viewpoint.

Application of Metabolic 13C Labeling in Conjunction with High-Field Nuclear Magnetic Resonance Spectroscopy for Comparative Conformational Analysis of High Mannose-Type Oligosaccharides.

High mannose-type oligosaccharides are enzymatically trimmed in the endoplasmic reticulum, resulting in various processing intermediates with exposed glycotopes that are recognized by a series of lectins involved in glycoprotein fate determination in cells. Although recent crystallographic data have provided the structural basis for the carbohydrate recognition of intracellular lectins, atomic information of dynamic oligosaccharide conformations is essential for a quantitative understanding of the energetics of carbohydrate-lectin interactions. Carbohydrate NMR spectroscopy is useful for characterizing such conformational dynamics, but often hampered by poor spectral resolution and lack of recombinant techniques required to produce homogeneous glycoforms. To overcome these difficulties, we have recently developed a methodology for the preparation of a homogeneous high mannose-type oligosaccharide with 13C labeling using a genetically engineered yeast strain. We herein successfully extended this method to result in the overexpression of 13C-labeled Man9GlcNAc2 (M9) with a newly engineered yeast strain with the deletion of four genes involved in N-glycan processing. This enabled high-field NMR analyses of 13C-labeled M9 in comparison with its processing product lacking the terminal mannose residue ManD2. Long-range NOE data indicated that the outer branches interact with the core in both glycoforms, and such foldback conformations are enhanced upon the removal of ManD2. The observed conformational variabilities might be significantly associated with lectins and glycan-trimming enzymes.

Identification and characterization of novel cell wall hydrolase CwlT: a two-domain autolysin exhibiting n-acetylmuramidase and DL-endopeptidase activities.

A cell wall hydrolase homologue, Bacillus subtilis YddH (renamed CwlT), was determined to be a novel cell wall lytic enzyme. The cwlT gene is located in the region of an integrative and conjugative element (ICEBs1), and a cwlT-lacZ fusion experiment revealed the significant expression when mitomycin C was added to the culture. Judging from the Pfam data base, CwlT (cell wall lytic enzyme T (Two-catalytic domains)) has two hydrolase domains that exhibit high amino acid sequence similarity to dl-endopeptidases and relatively low similarity to lytic transglycosylases at the C and N termini, respectively. The purified C-terminal domain of CwlT (CwlT-C-His) could hydrolyze the linkage of d-gamma-glutamyl-meso-diaminopimelic acid in B. subtilis peptidoglycan, suggesting that the C-terminal domain acts as a dl-endopeptidase. On the other hand, the purified N-terminal domain (CwlT-N-His) could also hydrolyze the peptidoglycan of B. subtilis. However, on reverse-phase HPLC and mass spectrometry (MS) and MS-MS analyses of the reaction products by CwlT-N-His, this domain was determined to act as an N-acetylmuramidase and not a lytic transglycosylase. Moreover, the site-directed mutagenesis analysis revealed that Glu-87 and Asp-94 are sites related with the cell wall lytic activity. Because the amino acid sequence of the N-terminal domain of CwlT exhibits low similarity compared with those of the soluble lytic transglycosylase and muramidase (goose lysozyme), this domain represents "a new category of cell wall hydrolases."

Characterization of new L,D-endopeptidase gene product CwlK (previous YcdD) that hydrolyzes peptidoglycan in Bacillus subtilis.

Bacillus subtilis has various cell wall hydrolases, however, the functions and hydrolase activities of some enzymes are still unknown. B. subtilis CwlK (YcdD) exhibits high sequence similarity with the peptidoglycan hydrolytic L,D-endopeptidase (PLY500) of Listeria monocytogenes phage and CwlK has the VanY motif which is a D-alanyl-D-alanine carboxypeptidase (Pfam: http://www.sanger.ac.uk/Software/Pfam/). The beta-galactosidase activity observed on cwlK-lacZ fusion indicated that the cwlK gene was expressed during the vegetative growth phase, and Western blotting suggested that CwlK seems to be localized in the membrane. Truncated CwlK fused with a histidine-tag (h-DeltaCwlK) was produced in Escherichia coli and purified on a nickel column. The h-DeltaCwlK protein hydrolyzed the peptidoglycan of B. subtilis, and the optimal pH, temperature and NaCl concentration for h-DeltaCwlK were pH 6.5, 37 degrees C, and 0 M, respectively. Interestingly, h-DeltaCwlK could hydrolyze the linkage of L-alanine-D-glutamic acid in the stem of the peptidoglycan, however, this enzyme could not hydrolyze the linkage of D-alanine-D-alanine, suggesting that CwlK is an L,D-endopeptidase not a D,D-carboxypeptidase. CwlK could not hydrolyze polyglutamate from B. natto or peptidoglycan of Staphylococcus aureus. This is the first report describing the characterization of an L,D-endopeptidase in B. subtilis and also the first report in bacteria of the characterization of a PLY500 family protein encoded in chromosomal DNA.

A polysaccharide deacetylase homologue, PdaA, in Bacillus subtilis acts as an N-acetylmuramic acid deacetylase in vitro.

A polysaccharide deacetylase homologue, PdaA, was determined to act as an N-acetylmuramic acid deacetylase in vitro. Histidine-tagged truncated PdaA (with the putative signal sequence removed) was overexpressed in Escherichia coli cells and purified. Measurement of deacetylase activity showed that PdaA could deacetylate peptidoglycan treated with N-acetylmuramoyl-L-alanine amidase CwlH but could not deacetylate peptidoglycan treated with or without DL-endopeptidase LytF (CwlE). Reverse-phase high-performance liquid chromatography and mass spectrometry (MS) and MS-MS analyses indicated that PdaA could deacetylate the N-acetylmuramic acid residues of purified glycan strands derived from Bacillus subtilis peptidoglycan.