Genetic and functional diversity of ß-N-acetylgalactosamine-targeting glycosidases expanded by deep-sea metagenome analysis.

ß-N-Acetylgalactosamine-containing glycans play essential roles in several biological processes, including cell adhesion, signal transduction, and immune responses. ß-N-Acetylgalactosaminidases hydrolyze ß-N-acetylgalactosamine linkages of various glycoconjugates. However, their biological significance remains ambiguous, primarily because only one type of enzyme, exo-ß-N-acetylgalactosaminidases that specifically act on ß-N-acetylgalactosamine residues, has been documented to date. In this study, we identify four groups distributed among all three domains of life and characterize eight ß-N-acetylgalactosaminidases and ß-N-acetylhexosaminidase through sequence-based screening of deep-sea metagenomes and subsequent searching of public protein databases. Despite low sequence similarity, the crystal structures of these enzymes demonstrate that all enzymes share a prototype structure and have diversified their substrate specificities (oligosaccharide-releasing, oligosaccharide/monosaccharide-releasing, and monosaccharide-releasing) through the accumulation of mutations and insertional amino acid sequences. The diverse ß-N-acetylgalactosaminidases reported in this study could facilitate the comprehension of their structures and functions and present evolutionary pathways for expanding their substrate specificity.

Development of a rapid and highly accurate method for 13C tracer-based metabolomics and its application on a hydrogenotrophic methanogen.

Microfluidic capillary electrophoresis-mass spectrometry (CE-MS) is a rapid and highly accurate method to determine isotopomer patterns in isotopically labeled compounds. Here, we developed a novel method for tracer-based metabolomics using CE-MS for underivatized proteinogenic amino acids. The method consisting of a ZipChip CE system and a high-resolution Orbitrap Fusion Tribrid mass spectrometer allows us to obtain highly accurate data from 1 µl of 100 nmol/l amino acids comparable to a mere 1 [Formula: see text] 104-105 prokaryotic cells. To validate the capability of the CE-MS method, we analyzed 16 protein-derived amino acids from a methanogenic archaeon Methanothermobacter thermautotrophicus as a model organism, and the mass spectra showed sharp peaks with low mass errors and background noise. Tracer-based metabolome analysis was then performed to identify the central carbon metabolism in M. thermautotrophicus using 13C-labeled substrates. The mass isotopomer distributions of serine, aspartate, and glutamate revealed the occurrence of both the Wood-Ljungdahl pathway and an incomplete reductive tricarboxylic acid cycle for carbon fixation. In addition, biosynthesis pathways of 15 amino acids were constructed based on the mass isotopomer distributions of the detected protein-derived amino acids, genomic information, and public databases. Among them, the presence of alternative enzymes of alanine dehydrogenase, ornithine cyclodeaminase, and homoserine kinase was suggested in the biosynthesis pathways of alanine, proline, and threonine, respectively. To our knowledge, the novel 13C tracer-based metabolomics using CE-MS can be considered the most efficient method to identify central carbon metabolism and amino acid biosynthesis pathways and is applicable to any kind of isolated microbe.

Diverse DNA modification in marine prokaryotic and viral communities.

DNA chemical modifications, including methylation, are widespread and play important roles in prokaryotes and viruses. However, current knowledge of these modification systems is severely biased towards a limited number of culturable prokaryotes, despite the fact that a vast majority of microorganisms have not yet been cultured. Here, using single-molecule real-time sequencing, we conducted culture-independent 'metaepigenomic' analyses (an integrated analysis of metagenomics and epigenomics) of marine microbial communities. A total of 233 and 163 metagenomic-assembled genomes (MAGs) were constructed from diverse prokaryotes and viruses, respectively, and 220 modified motifs and 276 DNA methyltransferases (MTases) were identified. Most of the MTase genes were not genetically linked with the endonuclease genes predicted to be involved in defense mechanisms against extracellular DNA. The MTase-motif correspondence found in the MAGs revealed 10 novel pairs, 5 of which showed novel specificities and experimentally confirmed the catalytic specificities of the MTases. We revealed novel alternative specificities in MTases that are highly conserved in Alphaproteobacteria, which may enhance our understanding of the co-evolutionary history of the methylation systems and the genomes. Our findings highlight diverse unexplored DNA modifications that potentially affect the ecology and evolution of prokaryotes and viruses in nature.

Structural Basis of Leader Peptide Recognition in Lasso Peptide Biosynthesis Pathway.

Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) with a unique 3D-interlocked structure, in which an N-terminal macrolactam ring is threaded by a linear C-terminal part. The unique structure of lasso peptides is introduced into ribosomally translated precursor peptides by lasso peptide synthetase encompassing proteins B and C or B1, B2, and C when the B enzyme is split into two distinct proteins. The B1 protein recognizes the leader sequence of the precursor peptide, and then the B2 protein cleaves it. The C protein catalyzes the formation of the macrolactam ring. However, the detailed mechanism of lasso peptide maturation has remained elusive, due to the lack of structural information about the responsible proteins. Here we report the crystal structure of the B1 protein from the thermophilic actinobacteria, Thermobifida fusca (TfuB1), complexed with the leader peptide (TfuA-Leader), which revealed the detailed mechanism of leader peptide recognition. The structure of TfuB1 consists of an N-terminal ß-sheet and three C-terminal helices. The leader peptide is docked on one edge of the N-terminal ß-sheet of TfuB1, as an additional ß strand. Three conserved amino acid residues of the leader peptide (TfuA Tyr-17, Pro-14, and Leu-12) fit well on the hydrophobic cleft between the ß-sheet and adjacent helices. Biochemical analysis demonstrated that these conserved residues are essential for affinity between TfuB1 and the TfuA-Leader. Furthermore, we found that TfuB1 and the leader peptide jointly form a hydrophobic patch on the ß-sheet, which includes the highly conserved TfuA Phe-6 and TfuB1 Tyr33. Homology modeling and mutational analysis of the B1 protein from a firmicute, Bacillus pseudomycoides (PsmB1), revealed that the hydrophobic patch is conserved in a wide range of species and involved in the cleavage activity of the B2 protein, indicating it forms the interaction surface for the B2 protein or the core part of the precursor peptide.

Fluorimetric assays for N-acetylgalactosamine-6-sulfatase and arylsulfatase B based on the natural substrates for confirmation of mucopolysaccharidoses types IVA and VI.

BACKGROUND: Treatments have been developed for mucopolysaccharidoses IVA (MPS IVA) and MPS VI suggesting the need for eventual newborn screening. Biochemical enzyme assays are important for diagnosis. Previously reported fluorimetric assays of the relevant enzymes are based on substrates with poor activity or specificity. METHODS: We developed new fluorimetric assays for N-acetylgalactosamine-6-sulfatase (GALNS) and arylsulfatase B (ARSB) based on the natural substrates, N-acetylgalactosamine-6-sulfate (and 4-sulfate), which have improved activity and specificity toward the relevant enzymes. The new substrates were tested on dried blood spots on newborn screening cards, and assays showed acceptable linearity in response with the amount of enzyme present (using quality control samples). RESULTS: When tested on dried blood spots from random newborns and affected patients, the assays showed good discrimination between the 2 sample groups. CONCLUSIONS: The analytical range of the new fluorimetric assays, defined as the ratio of enzyme-dependent-to-enzyme-independent assay response, is likely to be insufficient to use these assays for newborn screening. Rather, these new fluorimetric assays should be useful in a diagnostic lab to confirm a diagnosis via biochemical enzyme testing.

Conserved neutralizing epitope at globular head of hemagglutinin in H3N2 influenza viruses.

UNLABELLED: Neutralizing antibodies that target the hemagglutinin of influenza virus either inhibit binding of hemagglutinin to cellular receptors or prevent the low-pH-induced conformational change in hemagglutinin required for membrane fusion. In general, the former type of antibody binds to the globular head formed by HA1 and has narrow strain specificity, while the latter type binds to the stem mainly formed by HA2 and has broad strain specificity. In the present study, we analyzed the epitope and function of a broadly neutralizing human antibody against H3N2 viruses, F005-126. The crystal structure of F005-126 Fab in complex with hemagglutinin revealed that the antibody binds to the globular head, spans a cleft formed by two hemagglutinin monomers in a hemagglutinin trimer, and cross-links them. It recognizes two peptide portions (sites L and R) and a glycan linked to asparagine at residue 285 using three complementarity-determining regions and framework 3 in the heavy chain. Binding of the antibody to sites L (residues 171 to 173, 239, and 240) and R (residues 91, 92, 270 to 273, 284, and 285) is mediated mainly by van der Waals contacts with the main chains of the peptides in these sites and secondarily by hydrogen bonds with a few side chains of conserved sequences in HA1. Furthermore, the glycan recognized by F005-126 is conserved among H3N2 viruses. F005-126 has the ability to prevent low-pH-induced conformational changes in hemagglutinin. The newly identified conserved epitope, including the glycan, should be immunogenic in humans and may induce production of broadly neutralizing antibodies against H3 viruses. IMPORTANCE: Antibodies play an important role in protection against influenza virus, and hemagglutinin is the major target for virus neutralizing antibodies. It has long been believed that all effective neutralizing antibodies bind to the surrounding regions of the sialic acid-binding pocket and inhibit the binding of hemagglutinin to the cellular receptor. Since mutations are readily introduced into such epitopes, this type of antibody shows narrow strain specificity. Recently, however, broadly neutralizing antibodies have been isolated. Most of these bind either to conserved sites in the stem region or to the sialic acid-binding pocket itself. In the present study, we identified a new neutralizing epitope in the head region recognized by a broadly neutralizing human antibody against H3N2. This epitope may be useful for design of vaccines.

A novel crystal form of pyrrolysyl-tRNA synthetase reveals the pre- and post-aminoacyl-tRNA synthesis conformational states of the adenylate and aminoacyl moieties and an asparagine residue in the catalytic site.

Structures of Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS) have been determined in a novel crystal form. The triclinic form crystals contained two PylRS dimers (four monomer molecules) in the asymmetric unit, in which the two subunits in one dimer each bind N(ℇ)-(tert-butyloxycarbonyl)-L-lysyladenylate (BocLys-AMP) and the two subunits in the other dimer each bind AMP. The BocLys-AMP molecules adopt a curved conformation and the C(α) position of BocLys-AMP protrudes from the active site. The ß7-ß8 hairpin structures in the four PylRS molecules represent distinct conformations of different states of the aminoacyl-tRNA synthesis reaction. Tyr384, at the tip of the ß7-ß8 hairpin, moves from the edge to the inside of the active-site pocket and adopts multiple conformations in each state. Furthermore, a new crystal structure of the BocLys-AMPPNP-bound form is also reported. The bound BocLys adopts an unusually bent conformation, which differs from the previously reported structure. It is suggested that the present BocLys-AMPPNP-bound, BocLys-AMP-bound and AMP-bound complexes represent the initial binding of an amino acid (or pre-aminoacyl-AMP synthesis), pre-aminoacyl-tRNA synthesis and post-aminoacyl-tRNA synthesis states, respectively. The conformational changes of Asn346 that accompany the aminoacyl-tRNA synthesis reaction have been captured by X-ray crystallographic analyses. The orientation of the Asn346 side chain, which hydrogen-bonds to the carbonyl group of the amino-acid substrate, shifts by a maximum of 85-90° around the C(ß) atom.

Gaining insight into the inhibition of glycoside hydrolase family 20 exo-ß-N-acetylhexosaminidases using a structural approach.

One useful methodology that has been used to give insight into how chemically synthesized inhibitors bind to enzymes and the reasons underlying their potency is crystallographic studies of inhibitor-enzyme complexes. Presented here is the X-ray structural analysis of a representative family 20 exo-ß-N-acetylhexosaminidase in complex with various known classes of inhibitor of these types of enzymes, which highlights how different inhibitor classes can inhibit the same enzyme. This study will aid in the future development of inhibitors of not only exo-ß-N-acetylhexosaminidases but also other types of glycoside hydrolases.

Molecular cloning and catalytic mechanism of a novel glycosphingolipid-degrading beta-N-acetylgalactosaminidase from Paenibacillus sp. TS12.

We report here the molecular cloning, characterization, and catalytic mechanism of a novel glycosphingolipid-degrading ß-N-acetylgalactosaminidase (ß-NGA) from Paenibacillus sp. TS12 (NgaP). Consisting of 1034 putative amino acid residues, NgaP shares no sequence similarity with known proteins. Recombinant NgaP, expressed in Escherichia coli, cleaved the nonreducing terminal ß-GalNAc residues of gangliotriaosylceramide and globotetraosylceramide. The enzyme hydrolyzed para-nitrophenyl-ß-N-acetylgalactosaminide ∼100 times faster than para-nitrophenyl-ß-N-acetylglucosaminide. GalNAc thiazoline, an analog of the oxazolinium intermediate and potent inhibitor for enzymes adopting substrate-assisted catalysis, competitively inhibited the enzyme. The K(i) of the enzyme for GalNAc thiazoline was 1.3 nM, whereas that for GlcNAc thiazoline was 46.8 µM. Comparison of the secondary structure with those of known enzymes exhibiting substrate-assisted catalysis and point mutation analysis indicated that NgaP adopts substrate-assisted catalysis in which Glu-608 and Asp-607 could function as a proton donor and a stabilizer of the 2-acetamide group of the ß-GalNAc at the active site, respectively. These results clearly indicate that NgaP is a ß-NGA showing substrate-assisted catalysis. This is the first report describing the molecular cloning of a ß-NGA adopting substrate-assisted catalysis.

Crystallization and preliminary X-ray crystallographic study of GenX, a lysyl-tRNA synthetase paralogue from Escherichia coli, in complex with translation elongation factor P.

GenX, a lysyl-tRNA synthetase paralogue from Escherichia coli, was overexpressed in E. coli, purified by three chromatographic steps and cocrystallized with a lysyl adenylate analogue (LysAMS) by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. The GenX-LysAMS crystals belonged to the triclinic space group P1, with unit-cell parameters a=54.80, b=69.15, c=94.08 A, alpha=95.47, beta=106.51, gamma=90.46 degrees, and diffracted to 1.9 A resolution. Furthermore, GenX was cocrystallized with translation elongation factor P (EF-P), which is believed to be a putative substrate of GenX, and LysAMS using PEG 4000 and ammonium sulfate as precipitants. The GenX-EF-P-LysAMS crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a=105.93, b=102.96, c=119.94 A, beta=99.4 degrees, and diffracted to 2.5 A resolution. Structure determination of the E. coli GenX-LysAMS and GenX-EF-P-LysAMS complexes by molecular replacement was successful and structure refinements are now in progress.

A paralog of lysyl-tRNA synthetase aminoacylates a conserved lysine residue in translation elongation factor P.

Aminoacyl-tRNA synthetase (aaRS) paralogs with unknown functions exist in various species. We now report novel 'protein lysylation' by an Escherichia coli lysyl-tRNA synthetase paralog, GenX/PoxA/YjeA. X-ray crystallographic analysis shows that the structure of the GenX protein resembles that of a class II aaRS. Further in vitro studies reveal that it specifically aminoacylates EF-P with lysine. The shape of the protein substrate mimics that of the L-shaped tRNA, and its lysylation site corresponds to the tRNA 3' end. Thus, we show how the aaRS architecture can be adapted to achieve aminoacylation of a specific protein. Moreover, in vivo analyses reveal that the translation elongation factor P (EF-P) lysylation by GenX is enhanced by YjeK (lysine 2,3-aminomutase paralog), which is encoded next to the EF-P gene, and might convert alpha-lysyl-EF-P to beta-lysyl-EF-P. In vivo analyses indicate that the EF-P modification by GenX and YjeK is essential for cell survival.

Modeling of tRNA-assisted mechanism of Arg activation based on a structure of Arg-tRNA synthetase, tRNA, and an ATP analog (ANP).

The ATP-pyrophosphate exchange reaction catalyzed by Arg-tRNA, Gln-tRNA and Glu-tRNA synthetases requires the assistance of the cognate tRNA. tRNA also assists Arg-tRNA synthetase in catalyzing the pyrophosphorolysis of synthetic Arg-AMP at low pH. The mechanism by which the 3'-end A76, and in particular its hydroxyl group, of the cognate tRNA is involved with the exchange reaction catalyzed by those enzymes has yet to be established. We determined a crystal structure of a complex of Arg-tRNA synthetase from Pyrococcus horikoshii, tRNA(Arg)(CCU) and an ATP analog with Rfactor = 0.213 (Rfree = 0.253) at 2.0 A resolution. On the basis of newly obtained structural information about the position of ATP bound on the enzyme, we constructed a structural model for a mechanism in which the formation of a hydrogen bond between the 2'-OH group of A76 of tRNA and the carboxyl group of Arg induces both formation of Arg-AMP (Arg + ATP --> Arg-AMP + pyrophosphate) and pyrophosphorolysis of Arg-AMP (Arg-AMP + pyrophosphate --> Arg + ATP) at low pH. Furthermore, we obtained a structural model of the molecular mechanism for the Arg-tRNA synthetase-catalyzed deacylation of Arg-tRNA (Arg-tRNA + AMP --> Arg-AMP + tRNA at high pH), in which the deacylation of aminoacyl-tRNA bound on Arg-tRNA synthetase and Glu-tRNA synthetase is catalyzed by a quite similar mechanism, whereby the proton-donating group (-NH-C+(NH2)2 or -COOH) of Arg and Glu assists the aminoacyl transfer from the 2'-OH group of tRNA to the phosphate group of AMP at high pH.

Molecular cloning and crystal structural analysis of a novel beta-N-acetylhexosaminidase from Paenibacillus sp. TS12 capable of degrading glycosphingolipids.

We report the molecular cloning and characterization of two novel beta-N-acetylhexosaminidases (beta-HEX, EC 3.2.1.52) from Paenibacillus sp. strain TS12. The two beta-HEXs (Hex1 and Hex2) were 70% identical in primary structure, and the N-terminal region of both enzymes showed significant similarity with beta-HEXs belonging to glycoside hydrolase family 20 (GH20). Interestingly, however, the C-terminal region of Hex1 and Hex2 shared no sequence similarity with the GH20 beta-HEXs or other known proteins. Both recombinant enzymes, expressed in Escherichia coli BL21(DE3), hydrolyzed the beta-N-acetylhexosamine linkage of chitooligosaccharides and glycosphingolipids such as asialo GM2 and Gb4Cer in the absence of detergent. However, the enzyme was not able to hydrolyze GM2 ganglioside in the presence or in the absence of detergent. We determined three crystal structures of Hex1; the Hex1 deletion mutant Hex1-DeltaC at a resolution of 1.8 A; Hex1-DeltaC in complex with beta-N-acetylglucosamine at 1.6 A; and Hex1-DeltaC in complex with beta-N-acetylgalactosamine at 1.9 A. We made a docking model of Hex1-DeltaC with GM2 oligosaccharide, revealing that the sialic acid residue of GM2 could hinder access of the substrate to the active site cavity. This is the first report describing the molecular cloning, characterization and X-ray structure of a procaryotic beta-HEX capable of hydrolyzing glycosphingolipids.

Brain germinoma in a young adult man with suspicion of fatal neglect.

We report a forensic autopsy case of brain germinoma in a 26-year-old man who was severely wasted and initially suspected of fatal neglect. He had a history of nocturnal wandering and was confined by his parents. Neuropathological examination showed germinoma in the hypothalamus and neurohypophysis. The cause of death was certified as hypothalamo-hypophyseal insufficiency due to germinoma. Because hypothalamic lesions may dysregulate feeding behavior and sleeping rhythms, germinoma was considered the causative lesion of the anorexic wasting and nocturnal wandering. Confinement was a preventive measure of the patient's wandering. The findings in this case indicated that hypothalamic tumors should be recognized as a cause of wasting in autopsies suspected of fatal neglect.

Dementing diseases among elderly persons who suffered fatal accidents: a forensic autopsy study.

The demographics and forensic autopsy findings of 125 elderly persons were analyzed to identify the risk factors of fatal accidents among elderly and to develop preventive measures to minimize such events. Cliniconeuropathologic dementing diseases were diagnosed in 13 of the 69 accidental death but only 1 of the 56 nonaccidental death cases, indicating that dementing diseases are associated with accidental deaths of elderly in forensic autopsy populations and suggesting that interventions for preventing fatal accidents should focus on elderly persons with dementia. Blood alcohol was only detected in persons without dementia, indicating that dementing diseases and drunkenness are not coexisting factors for fatal accidents among elderly. Living alone might increase the risk of mortalities associated with accidental injuries because of the absence of a caregiver at the scene and delayed medical help. The majority of fatal accidents occurred outdoors, emphasizing the need for interventions to reduce environmental hazards such as those related to traffic, open water, and cold weather. Increased public awareness of accident risks and preventive interventions will reduce accidental deaths among community-dwelling elderly people.

Unexpected death due to intestinal obstruction by a duplication cyst in an infant.

An 11-month-old female infant died at home after being diagnosed as having gastroenteritis. Autopsy examination revealed a distended distal ileum filled with a large amount of watery contents, and a 3 cm x 3 cm x 4 cm cyst at the ileocecal part of the cecum that was histologically consistent with a duplication cyst. The cause of death was determined to be acute dehydration due to intestinal obstruction caused by a duplication cyst. This case indicates that intestinal obstruction by a duplication cyst should be recognized as a cause of unexpected death in infants.

Dural hemorrhage of the tentorium on postmortem cranial computed tomographic scans in children.

We analyzed four children with increased density of the tentorium on postmortem computed tomographic (CT) scans that suggested traumatic subarachnoid hemorrhage; but in which followup autopsy demonstrated an absence of subarachnoid hemorrhage. All cases had neither head injuries nor distinct intracranial abnormalities. Histological examinations showed extensive hemorrhage within the tentorium. These findings indicate that increased density of the tentorium on postmortem CT scan reflects 'dural hemorrhage' that may occur in children without intracranial abnormalities. Pediatricians and forensic pathologists should be aware of this phenomenon when using postmortem preautopsy CT scanning for diagnosis of child abuse.

Utilization of ganglioside-degrading Paenibacillus sp. strain TS12 for production of glucosylceramide.

Gangliosides, sialic acid-containing glycosphingolipids, are membrane constituents of vertebrates and are known to have important roles in cellular differentiation, adhesion, and recognition. We report here the isolation of a bacterium capable of degrading gangliotetraose-series gangliosides and a new method for the production of glucosylceramide with this bacterium. GM1a ganglioside was found to be sequentially degraded by Paenibacillus sp. strain TS12, which was isolated from soil, as follows: GM1a --> asialo GM1 --> asialo GM2 --> lactosylceramide --> glucosylceramide. TS12 was found to produce a series of ganglioside-degrading enzymes, such as sialidases, beta-galactosidases, and beta-hexosaminidases. TS12 also produced beta-glucosidases, but glucosylceramide was somewhat resistant to the bacterial enzyme under the conditions used. Taking advantage of the specificity, we developed a new method for the production of glucosylceramide using TS12 as a biocatalyst. The method involves the conversion of crude bovine brain gangliosides to glucosylceramide by coculture with TS12 and purification of the product by chromatography with Wakogel C-300 HG.

Molecular cloning and characterization of a novel glucocerebrosidase of Paenibacillus sp. TS12.

We report here the molecular cloning and characterization of a glucocerebrosidase [EC 3.2.1.45] from Paenibacillus sp. TS12. The open reading frame of the glucocerebrosidase gene consisted of 2,493 bp nucleotides and encoded 831 amino acid residues. The enzyme exhibited no sequence similarity with a classical glucocerebrosidase belonging to glycoside hydrolase (GH) family 30, but rather showed significant similarity with GH family 3 beta-glucosidases from Clostridium thermocellum, Ruminococcus albus, and Aspergillus aculeateus. The recombinant enzyme, expressed in Escherichia coli BL21(DE3)pLysS, had a molecular weight of 90.7 kDa and hydrolyzed NBD-labeled glucosylceramide, but not galactosylceramide, GM1a or sphingomyelin. The enzyme was most active at pH 6.5, and its apparent Km and Vmax values for NBD-labeled glucosylceramide and p-nitrophenyl-beta-glucopyranoside were 223 microM and 1.60 micromol/min/mg of protein, and 593 microM and 112 micromol/min/mg of protein, respectively. Site-directed mutagenesis indicated that Asp-223 is an essential amino acid for the catalytic reaction and possibly functions a catalytic nucleophile, as in GH family 3 beta-glucosidases. This is the first report of the molecular cloning and characterization of a glucocerebrosidase from a procaryote.