O-Glycosylation as a Novel Control Mechanism of Peptidoglycan Hydrolase Activity

Acm2, the major autolysin of Lactobacillus plantarum, is a tripartite protein. Its catalytic domain is surrounded by an O-glycosylated N-terminal region rich in Ala, Ser, and Thr (AST domain), which is of low complexity and unknown function, and a C-terminal region composed of five SH3b peptidoglycan (PG) binding domains. Here, we investigate the contribution of these two accessory domains and of O-glycosylation to Acm2 functionality. We demonstrate that Acm2 is an N-acetylglucosaminidase and identify the pattern of O-glycosylation (21 mono-N-acetylglucosamines) of its AST domain. The O-glycosylation process is species-specific as Acm2 purified from Lactococcus lactis is not glycosylated. We therefore explored the functional role of O-glycosylation by purifying different truncated versions of Acm2 that were either glycosylated or non-glycosylated. We show that SH3b domains are able to bind PG and are responsible for Acm2 targeting to the septum of dividing cells, whereas the AST domain and its O-glycosylation are not involved in this process. Notably, our data reveal that the lack of O-glycosylation of the AST domain significantly increases Acm2 enzymatic activity, whereas removal of SH3b PG binding domains dramatically reduces this activity. Based on this antagonistic role, we propose a model in which access of the Acm2 catalytic domain to its substrate may be hindered by the AST domain where O-glycosylation changes its conformation and/or mediates interdomain interactions. To the best of our knowledge, this is the first time that O-glycosylation is shown to control the activity of a bacterial enzyme.     

Crystal Structure of the Catalytic Domain of the Bacillus cereus SleB Protein, Important in Cortex Peptidoglycan Degradation during Spore Germination

The SleB protein is one of two redundant cortex-lytic enzymes (CLEs) that initiate the degradation of cortex peptidoglycan (PG), a process essential for germination of spores of Bacillus species, including Bacillus anthracis. SleB has been characterized as a soluble lytic transglycosylase that specifically recognizes spore cortex PG and catalyzes the cleavage of glycosidic bonds between N-acetylmuramic acid (NAM) and N-acetylglucosamine residues with concomitant formation of a 1,6-anhydro bond in the NAM residue. We found that like the full-length Bacillus cereus SleB, the catalytic C-terminal domain (SleB^C) exhibited high degradative activity on cortex PG in vitro, although SleB''s N-terminal domain, thought to bind PG, was inactive. The 1.85-Å crystal structure of SleB^C reveals an ellipsoid molecule with two distinct domains dominated by either α helices or β strands. The overall fold of SleB closely resembles that of the catalytic domain of the family 1 lytic transglycosylases but with a completely different topological arrangement. Structural analysis shows that an invariant Glu157 of SleB is in a position equivalent to that of the catalytic glutamate in other lytic transglycosylases. Indeed, SleB bearing a Glu157-to-Gln mutation lost its cortex degradative activity completely. In addition, the other redundant CLE (called CwlJ) in Bacillus species likely has a three-dimensional structure similar to that of SleB, including the invariant putative catalytic Glu residue. SleB and CwlJ may offer novel targets for the development of anti-spore agents.     

Specific Structural Features of the N-Acetylmuramoyl-l-Alanine Amidase AmiD from Escherichia coli and Mechanistic Implications for Enzymes of This Family

AmiD is the fifth identified N-acetylmuramoyl-l-alanine zinc amidase of Escherichia coli. This periplasmic lipoprotein is anchored in the outer membrane and has a broad specificity. AmiD is capable of cleaving the intact peptidoglycan (PG) as well as soluble fragments containing N-acetylmuramic acid regardless of the presence of an anhydro form or not, unlike the four other amidases, AmiA, AmiB, AmiC, and AmpD, which have some specificity. AmiD function is, however, not clearly established but it could be part of the enzymatic machinery involved in the PG turnover in E. coli. We solved three structures of the E. coli zinc amidase AmiD devoid of its lipidic anchorage: the holoenzyme, the apoenzyme in complex with the substrate anhydro-N-acetylmuramic-acid-l-Ala-γ-d-Glu-l-Lys, and the holoenzyme in complex with the l-Ala-γ-d-Glu-l-Lys peptide, the product of the hydrolysis of this substrate by AmiD. The AmiD structure shows a relatively flexible N-terminal extension that allows an easy reach of the PG by the enzyme inserted into the outer membrane. The C-terminal domain provides a potential extended geometrical complementarity to the substrate. AmiD shares a common fold with AmpD, the bacteriophage T7 lysozyme, and the PG recognition proteins, which are receptor proteins involved in the innate immune responses of a wide range of organisms. Analysis of the different structures reveals the similarity between the catalytic mechanism of zinc amidases of the AmiD family and the thermolysin-related zinc peptidases.     

NMR structural analysis of the soluble domain of ZiaA-ATPase and the basis of selective interactions with copper metallochaperone Atx1

A Cu(I) metallochaperone, Atx1, interacts with the amino-terminal domain of a Cu(I)-transporting ATPase, PacS_N, but not with a domain of related Zn-transporting ATPase, ZiaA_N in Synechocystis PCC 6803. This is thought to prevent ZiaA_N from acquiring Cu(I), which it binds more tightly than Zn. Solution structures of Atx1, PacS_N, and the heterodimer were previously described. Here we report solution structural studies of the ZiaA_N soluble domain. Apo-ZiaA_N has a typical ferredoxin-like fold followed by an atypical 34 residues of unstructured polypeptide containing a His₇ motif. ZiaA_N competes with the metallochromic indicator 4-(2-pyridylazo)resorcinol for 1 equiv of Zn, which can be displaced by thiol-modifying p-mercuriphenylsulfonic acid, establishing that a high-affinity site involves thiols of the CXXC motif within the ferredoxin-like fold. A single equivalent of Zn affects nuclear magnetic resonance signals arising from the CXXC motif as well as all seven His residues. The presence of NMR-line broadening in both sites implies that Zn₁-ZiaA_N undergoes exchange phenomena, consistent with CXXC-bound Zn coincidentally sampling various His ligands. These Zn-dependent dynamic changes could either aid metal transfer or alter intramolecular interactions. No formation of Atx1–Cu(I)–ZiaA_N heterodimers was observed, and in the presence of equimolar ZiaA_N and PacS_N, only Atx1–Cu(I)–PacS_N complexes were detected. Residues flanking the CXXC motif of PacS_N (R₁₃-ASS₂₀) differ in charge and bulk from those of ZiaA_N (D₁₈-KLK₂₅) and make contacts in the Atx1–Cu(I)–PacS_N complex. Crucially, swapping these residues flanking the CXXC motifs of ZiaA_N and PacS_N reciprocally swaps partner choice by Atx1. These few residues of the two ATPases have diverged during evolution to bias Atx1 interactions in favor of PacS_N rather than ZiaA_N.     

Structure-function analysis of yeast RNA debranching enzyme (Dbr1), a manganese-dependent phosphodiesterase

Saccharomyces cerevisiae Dbr1 is a 405-amino acid RNA debranching enzyme that cleaves the 2′-5′ phosphodiester bonds of the lariat introns formed during pre-mRNA splicing. Debranching appears to be a rate-limiting step for the turnover of intronic RNA, insofar as the steady-state levels of lariat introns are greatly increased in a Δdbr1 strain. To gain insight to the requirements for yeast Dbr1 function, we performed a mutational analysis of 28 amino acids that are conserved in Dbr1 homologs from other organisms. We identified 13 residues (His13, Asp40, Arg45, Asp49, Tyr68, Tyr69, Asn85, His86, Glu87, His179, Asp180, His231 and His233) at which alanine substitutions resulted in lariat intron accumulation in vivo. Conservative replacements at these positions were introduced to illuminate structure–activity relationships. Residues important for Dbr1 function include putative counterparts of the amino acids that comprise the active site of the metallophosphoesterase superfamily, exemplified by the DNA phosphodiesterase Mre11. Using natural lariat RNAs and synthetic branched RNAs as substrates, we found that mutation of Asp40, Asn85, His86, His179, His231 or His233 to alanine abolishes or greatly diminishes debranching activity in vitro. Dbr1 sediments as a monomer and requires manganese as the metal cofactor for debranching.     

The role of asparagine-linked glycosylation site on the catalytic domain of matriptase in its zymogen activation

Matriptase is a type II transmembrane serine protease containing one potential site for asparagine-linked glycosylation (N-glycosylation) on the catalytic domain (Asn772). It has been found that the activation of matriptase zymogen occurs via a mechanism requiring its own activity and that the N-glycosylation site is critical for the activation. The present study aimed to determine the underlying reasons for the site requirement using Madin–Darby canine kidney cells stably expressing recombinant variants of rat matriptase. A full-length variant with glutamine substitution at Asn772 appeared to be unable to undergo activation because of its catalytic incompetence (i.e., decreased availability of the soluble catalytic domain and/or of the correctly folded domain). This was evidenced by the observations that (i) a recombinant catalytic domain of matriptase with glutamine substitution at the site corresponding to matriptase Asn772 [N772Q-CD-Myc(His)₆] was not detected in the medium conditioned by transfected cells but was on the cell surface and (ii) purified N772Q-CD-Myc(His)₆ exhibited markedly reduced activity toward a peptide substrate. It is concluded that N-glycosylation site at Asn772 of matriptase is required for the zymogen activation because it plays an important role in rendering this protease catalytically competent in the cellular environment.     

Biochemical and structural characterization of a novel family of cystathionine beta-synthase domain proteins fused to a Zn ribbon-like domain

We have identified a novel family of proteins, in which the N-terminal cystathionine β-synthase (CBS) domain is fused to the C-terminal Zn ribbon domain. Four proteins were overexpressed in Escherichia coli and purified: TA0289 from Thermoplasma acidophilum, TV1335 from Thermoplasma volcanium, PF1953 from Pyrococcus furiosus, and PH0267 from Pyrococcus horikoshii. The purified proteins had a red/purple color in solution and an absorption spectrum typical of rubredoxins (Rds). Metal analysis of purified proteins revealed the presence of several metals, with iron and zinc being the most abundant metals (2-67% of iron and 12-74% of zinc). Crystal structures of both mercury- and iron-bound TA0289 (1.5-2.0 Å resolution) revealed a dimeric protein whose intersubunit contacts are formed exclusively by the α-helices of two cystathionine β-synthase subdomains, whereas the C-terminal domain has a classical Zn ribbon planar architecture. All proteins were reversibly reduced by chemical reductants (ascorbate or dithionite) or by the general Rd reductase NorW from E. coli in the presence of NADH. Reduced TA0289 was found to be capable of transferring electrons to cytochrome C from horse heart. Likewise, the purified Zn ribbon protein KTI11 from Saccharomyces cerevisiae had a purple color in solution and an Rd-like absorption spectrum, contained both iron and zinc, and was reduced by the Rd reductase NorW from E. coli. Thus, recombinant Zn ribbon domains from archaea and yeast demonstrate an Rd-like electron carrier activity in vitro. We suggest that, in vivo, some Zn ribbon domains might also bind iron and therefore possess an electron carrier activity, adding another physiological role to this large family of important proteins.     

The Metalloregulatory Zinc Site in Streptococcus pneumoniae AdcR, a Zinc-activated MarR Family Repressor

Streptococcus pneumoniae D39 AdcR (adhesin competence repressor) is the first metal-sensing member of the MarR (multiple antibiotic resistance repressor) family to be characterized. Expression profiling with a ΔadcR strain grown in liquid culture (brain-heart infusion) under microaerobic conditions revealed upregulation of 13 genes, including adcR and adcCBA, encoding a high-affinity ABC uptake system for zinc, and genes encoding cell-surface zinc-binding pneumococcal histidine triad (Pht) proteins and AdcAII (Lmb, laminin binding). The ΔadcR, H108Q and H112Q adcR mutant allelic strains grown in 0.2 mM Zn(II) exhibit a slow-growth phenotype and an approximately twofold increase in cell-associated Zn(II). Apo- and Zn(II)-bound AdcR are homodimers in solution and binding to a 28-mer DNA containing an adc operator is strongly stimulated by Zn(II) with K_DNA-Zn = 2.4 × 10⁸ M^- 1 (pH 6.0, 0.2 M NaCl, 25 °C). AdcR binds two Zn(II) per dimer, with stepwise Zn(II) affinities K_Zn1 and K_Zn2 of ≥ 10⁹ M^- 1 at pH 6.0 and ≥ 10¹² M^- 1 at pH 8.0, and one to three lower affinity Zn(II) depending on the pH. X-ray absorption spectroscopy of the high-affinity site reveals a pentacoordinate N/O complex and no cysteine coordination, the latter finding corroborated by wild type-like functional properties of C30A AdcR. Alanine substitution of conserved residues His42 in the DNA-binding domain, and His108 and His112 in the C-terminal regulatory domain, abolish high-affinity Zn(II) binding and greatly reduce Zn(II)-activated binding to DNA. NMR studies reveal that these mutants adopt the same folded conformation as dimeric wild type apo-AdcR, but fail to conformationally switch upon Zn(II) binding. These studies implicate His42, His108 and H112 as metalloregulatory zinc ligands in S. pneumoniae AdcR.     

A second endolysin gene is fully embedded in-frame with the lysA gene of mycobacteriophage Ms6

Mycobacteriophages are dsDNA viruses that infect mycobacterial hosts. The mycobacteriophage Ms6 accomplishes lysis by producing two cell wall hydrolytic enzymes, Lysin A (LysA) that possesses a central peptidoglycan recognition protein (PGRP) super-family conserved domain with the amidase catalytic site, that cleaves the amide bond between the N-acetylmuramic acid and L-alanine residues in the oligopeptide crosslinking chains of the peptidoglycan and Lysin B (LysB) a mycolylarabinogalactan esterase that hydrolyzes the mycolic acids from the mycolyl-arabinogalactan-peptidoglycan complex. Examination of the endolysin (lysA) DNA sequence revealed the existence of an embedded gene (lysA(241)) encoded in the same reading frame and preceded by a consensus ribosome-binding site. In the present work we show that, even though lysA is essential for Ms6 viability, phage mutants that express only the longer (Lysin(384)) or the shorter (Lysin(241)) endolysin are viable, but defective in the normal timing, progression and completion of host cell lysis. In addition, both endolysins have peptidoglycan hydrolase activity and demonstrated broad growth inhibition activity against various gram-positive bacteria and mycobacteria.     

Structural and Biochemical Insights into the Peptidoglycan Hydrolase Domain of FlgJ from Salmonella typhimurium

FlgJ is a glycoside hydrolase (GH) enzyme belonging to the Carbohydrate Active enZyme (CAZy) family GH73. It facilitates passage of the bacterial flagellum through the peptidoglycan (PG) layer by cleaving the β-1,4 glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid sugars that comprise the glycan strands of PG. Here we describe the crystal structure of the GH domain of FlgJ from bacterial pathogen Salmonella typhimurium (StFlgJ). Interestingly, the active site of StFlgJ was blocked by the C-terminal α-helix of a neighbouring symmetry mate and a β-hairpin containing the putative catalytic glutamic acid residue Glu223 was poorly resolved and could not be completely modeled into the electron density, suggesting it is flexible. Previous reports have shown that the GH73 enzyme Auto from Listeria monocytogenes is inhibited by an N-terminal α-helix that may occlude the active site in similar fashion. To investigate if the C-terminus of StFlgJ inhibits GH activity, the glycolytic activity of StFlgJ was assessed with and without the C-terminal α-helix. The GH activity of StFlgJ was unaffected by the presence or absence of the α-helix, suggesting it is not involved in regulating activity. Removal of the C-terminal α-helix did, however, allow a crystal structure of the domain to be obtained where the flexible β-hairpin containing residue Glu223 was entirely resolved. The β-hairpin was positioned such that the active site groove was fully solvent-exposed, placing Glu223 nearly 21.6 Å away from the putative general acid/base residue Glu184, which is too far apart for these two residues to coordinate glycosidic bond hydrolysis. The mobile nature of the StFlgJ β-hairpin is consistent with structural studies of related GH73 enzymes, suggesting that a dynamic active site may be common to many GH73 enzymes, in which the active site opens to capture substrate and then closes to correctly orient active site residues for catalysis.     

Substrate specificity and structure-function analysis of the 3'-phosphoesterase component of the bacterial NHEJ protein, DNA ligase D

DNA ligase D (LigD) performs end remodeling and end sealing reactions during nonhomologous end joining in bacteria. Pseudomonas aeruginosa LigD consists of a central ATP-dependent ligase domain fused to a C-terminal polymerase domain and an N-terminal phosphoesterase (PE) module. The PE domain catalyzes manganese-dependent phosphodiesterase and phosphomonoesterase reactions at the 3' end of the primer strand of a primer-template. The phosphodiesterase cleaves a 3'-terminal diribonucleotide to yield a primer strand with a ribonucleoside 3'-PO4 terminus. The phosphomonoesterase converts a terminal ribonucleoside 3'-PO4 or deoxyribonucleoside 3'-PO4 of a primer-template to a 3'-OH. Here we report that the phosphodiesterase and phosphomonoesterase activities are both dependent on the presence and length of the 5' single-strand tail of the primer-template substrate. Although the phosphodiesterase activity is strictly dependent on the 2'-OH of the penultimate ribose, it is indifferent to a 2'-OH versus a2'-H on the terminal nucleoside. Incision at the ribonucleotide linkage is suppressed when the 2'-OH is moved by 1 nucleotide in the 5' direction, suggesting that LigD is an exoribonuclease that cleaves the 3'-terminal phosphodiester. We report the effects of conservative amino acid substitutions at residues: (i) His42, His48, Asp50, Arg52, His84, and Tyr88, which are essential for both the ribonuclease and 3'-phosphatase activities; (ii) Arg14, Asp15, Glu21, and Glu82, which are critical for 3'-phosphatase activity but not 3'-ribonucleoside removal; and (iii) at Lys66 and Arg76, which participate selectively in the 3'-ribonuclease reaction. The results suggest roles for individual functional groups in metal binding and/or phosphoesterase chemistry.     

Mycobacteriophage Lysin B is a novel mycolylarabinogalactan esterase

Mycobacteriophages encounter a unique problem among phages of Gram-positive bacteria, in that lysis must not only degrade the peptidoglycan layer but also circumvent a mycolic acid-rich outer membrane covalently attached to the arabinogalactan–peptidoglycan complex. Mycobacteriophages accomplish this by producing two lysis enzymes, Lysin A (LysA) that hydrolyses peptidoglycan, and Lysin B (LysB), a novel mycolylarabinogalactan esterase, that cleaves the mycolylarabinogalactan bond to release free mycolic acids. The D29 LysB structure shows an α/β hydrolase organization with a catalytic triad common to cutinases, but which contains an additional four-helix domain implicated in the binding of lipid substrates. Whereas LysA is essential for mycobacterial lysis, a Giles Δ lysB mutant mycobacteriophage is viable, but defective in the normal timing, progression and completion of host cell lysis. We propose that LysB facilitates lysis by compromising the integrity of the mycobacterial outer membrane linkage to the arabinogalactan–peptidoglycan layer.     

Characterization of the zinc binding activity of the rubella virus nonstructural protease

The rubella virus (RUB) nonstructural (NS) protein (NSP) ORF encodes a protease that cleaves the NSP precursor (240 kDa) at a single site to produce two products. A cleavage site mutation was introduced into a RUB infectious cDNA clone and found to be lethal, demonstrating that cleavage of the NSP precursor is necessary for RUB replication. Based on computer alignments, the RUB NS protease was predicted to be a papain-like cysteine protease (PCP) with the residues Cys1152 and His1273 as the catalytic dyad; however, the RUB NS protease was recently found to require divalent cations such as Zn, Co, and Cd for activity (X. Liu, S. L. Ropp, R. J. Jackson, and T. K. Frey, J. Virol. 72:4463-4466, 1998). To analyze the function of metal cation binding in protease activity, Zn binding studies were performed using the minimal NS protease domain within the NSP ORF. When expressed as a maltose binding protein (MBP) fusion protein by bacteria, the NS protease exhibited activity both in the bacteria and in vitro following purification when denatured and refolded in the presence of Zn. Atomic absorption analysis detected 1.6 mol of Zn bound per mol of protein refolded in this manner. Expression of individual domains within the protease as MBP fusions and analysis by a Zn(65) binding assay revealed two Zn binding domains: one located at a predicted metal binding motif beginning at Cys1175 and the other one close to the cleavage site. Mutagenesis studies showed that Cys1175 and Cys1178 in the first domain and Cys1227 and His1273, the His in the predicted catalytic site, in the second domain are essential for zinc binding. All of these residues are also necessary for the protease activity, as were several other Cys residues not involved in Zn binding. Far-UV circular dichroism (CD) analysis of the MBP-NS protease fusion protein showed that the protease domain contained a large amount of alpha-helical structure, which is consistent with the results of secondary-structural prediction. Both far-UV-CD and fluorescence studies suggested that Zn did not exert a major effect on the overall structure of the fusion protein. Finally, protease inhibitor assays found that the protease activity can be blocked by both metal ion chelators and the metalloprotease inhibitor captopril. In conjunction with the finding that the previously predicted catalytic site, His1273, is essential for zinc binding, this suggests that the RUB NS protease is actually a novel virus metalloprotease rather than a PCP.     

Growth Medium-Dependent Glycine Incorporation into the Peptidoglycan of Caulobacter crescentus

The peptidoglycan (PG) is a macromolecular component of the bacterial cell wall that maintains the shape and integrity of the cell. The PG of Caulobacter crescentus, unlike that of many other Gram-negative bacteria, has repeatedly been shown to contain significant amounts of glycine. This compositional peculiarity has been deemed an intrinsic characteristic of this species. By performing a comprehensive qualitative and quantitative analysis of the C. crescentus PG by high-performance liquid chromatography (HPLC) and mass spectrometry (MS), we show here that glycine incorporation into the C. crescentus PG depends on the presence of exogenous glycine in the growth medium. High levels of glycine were detected at the fifth position of the peptide side chains of PG isolated from C. crescentus cells grown in the complex laboratory medium PYE or in defined medium (M2G) supplemented with casamino acids or glycine alone. In contrast, glycine incorporation was undetectable when cells were grown in M2G medium lacking glycine. Remarkably, glycine incorporation into C. crescentus peptidoglycan occurred even in the presence of low millimolar to sub-millimolar concentrations of free glycine. High glycine content in the PG had no obvious effects on growth rates, mode of PG incorporation or cell morphology. Hence, the C. crescentus PG is able to retain its physiological functions in cell growth and morphogenesis despite significant alterations in its composition, in what we deem to be unprecedented plasticity.     

Archaeal JAB1/MPN/MOV34 metalloenzyme (HvJAMM1) cleaves ubiquitin‐like small archaeal modifier proteins (SAMPs) from protein‐conjugates

Proteins with JAB1/MPN/MOV34 metalloenzyme (JAMM/MPN+) domains are widespread among all domains of life, yet poorly understood. Here we report the purification and characterization of an archaeal JAMM/MPN+ domain protein (HvJAMM1) from Haloferax volcanii that cleaves ubiquitin‐like small archaeal modifier proteins (SAMP1/2) from protein conjugates. HvJAMM1 cleaved SAMP1/2 conjugates generated in H. volcanii as well as isopeptide‐ and linear‐linked SAMP1–MoaE in purified form. Cleavage of linear linked SAMP1–MoaE was dependent on the presence of the SAMP domain and the C‐terminal VSGG motif of this domain. While HvJAMM1 was inhibited by size exclusion chromatography and metal chelators, its activity could be restored by addition of excess ZnCl₂. HvJAMM1 residues (Glu31, His88, His90, Ser98 and Asp101) that were conserved with the JAMM/MPN+ active‐site motif were required for enzyme activity. Together, these results provide the first example of a JAMM/MPN+ zinc metalloprotease that independently catalyses the cleavage of ubiquitin‐like (isopeptide and linear) bonds from target proteins. In archaea, HvJAMM1 likely regulates sampylation and the pools of ‘free’ SAMP available for protein modification. HvJAMM1‐type proteins are thought to release the SAMPs from proteins modified post‐translationally as well as those synthesized as domain fusions.     

N-acetylglucosaminyltransferase-V requires a specific noncatalytic luminal domain for its activity toward glycoprotein substrates

N-acetylglucosaminyltransferase-V (GnT-V or MGAT5) catalyzes the formation of an N-glycan β1,6-GlcNAc branch on selective target proteins in the Golgi apparatus and is involved in cancer malignancy and autoimmune disease etiology. Several three-dimensional structures of GnT-V were recently solved, and the recognition mechanism of the oligosaccharide substrate was clarified. However, it is still unclear how GnT-V selectively acts on glycoprotein substrates. In this study, we focused on an uncharacterized domain at the N-terminal side of the luminal region (N domain) of GnT-V, which was previously identified in a crystal structure, and aimed to reveal its role in GnT-V action. Using lectin blotting and fluorescence assisted cell sorting analysis, we found that a GnT-VΔN mutant lacking the N domain showed impaired biosynthetic activity in cells, indicating that the N domain is required for efficient glycosylation. To clarify this mechanism, we measured the in vitro activity of purified GnT-VΔN toward various kinds of substrates (oligosaccharide, glycohexapeptide, and glycoprotein) using HPLC and a UDP-Glo assay. Surprisingly, GnT-VΔN showed substantially reduced activity toward the glycoprotein substrates, whereas it almost fully maintained its activity toward the oligosaccharides and the glycopeptide substrates. Finally, docking models of GnT-V with substrate glycoproteins suggested that the N domain could interact with the substrate polypeptide directly. Our findings suggest that the N domain of GnT-V plays a critical role in the recognition of glycoprotein substrates, providing new insights into the mechanism of substrate-selective biosynthesis of N-glycans.     

Binding of Zn2+ to a Ca2+ loop allosterically attenuates the activity of factor VIIa and reduces its affinity for tissue factor

The protease domain of coagulation factor VIIa (FVIIa) is homologous to trypsin with a similar active site architecture. The catalytic function of FVIIa is regulated by allosteric modulations induced by binding of divalent metal ions and the cofactor tissue factor (TF). To further elucidate the mechanisms behind these transformations, the effects of Zn2+ binding to FVIIa in the free form and in complex with TF were investigated. Equilibrium dialysis suggested that two Zn2+ bind with high affinity to FVIIa outside the N-terminal gamma-carboxyglutamic acid (Gla) domain. Binding of Zn2+ to FVIIa, which was influenced by the presence of Ca2+, resulted in decreased amidolytic activity and slightly reduced affinity for TF. After binding to TF, FVIIa was less susceptible to zinc inhibition. Alanine substitutions for either of two histidine residues unique for FVIIa, His216, and His257, produced FVIIa variants with decreased sensitivity to Zn2+ inhibition. A search for putative Zn2+ binding sites in the crystal structure of the FVIIa protease domain was performed by Grid calculations. We identified a pair of Zn2+ binding sites in the Glu210-Glu220 Ca2+ binding loop adjacent to the so-called activation domain canonical to serine proteases. Based on our results, we propose a model that describes the conformational changes underlying the Zn2+-mediated allosteric down-regulation of FVIIa's activity.     

Separation and characterization of endopolygalacturonase and exopolygalacturonase from peaches

Two polygalacturonases (PG I and PG II) have been separated from extracts of ripe peaches (Prunus persica) by chromatography on Sephadex G-100. PG I hydrolyzes polygalacturonic acid from the nonreducing ends of the molecules, releasing galacturonic acid as the product. It functions optimally at pH 5.5, requires Ca²⁺ for activity, and hydrolyzes low molecular weight substrates most rapidly. In contrast, PG II cleaves the molecular chain of the substrate randomly with a pH optimum at about 4. This enzyme is most reactive with substrates of intermediate molecular weight. It catalyzes the release of water-soluble, but 70% ethanol-insoluble, pectin from washed peach cell walls.     

A Novel Type of Peptidoglycan-binding Domain Highly Specific for Amidated d-Asp Cross-bridge,Identified in Lactobacillus casei Bacteriophage Endolysins

Peptidoglycan hydrolases (PGHs) are responsible for bacterial cell lysis. Most PGHs have a modular structure comprising a catalytic domain and a cell wall-binding domain (CWBD). PGHs of bacteriophage origin, called endolysins, are involved in bacterial lysis at the end of the infection cycle. We have characterized two endolysins, Lc-Lys and Lc-Lys-2, identified in prophages present in the genome of Lactobacillus casei BL23. These two enzymes have different catalytic domains but similar putative C-terminal CWBDs. By analyzing purified peptidoglycan (PG) degradation products, we showed that Lc-Lys is an N-acetylmuramoyl-l-alanine amidase, whereas Lc-Lys-2 is a γ-d-glutamyl-l-lysyl endopeptidase. Remarkably, both lysins were able to lyse only Gram-positive bacterial strains that possess PG with d-Ala⁴→d-Asx-l-Lys³ in their cross-bridge, such as Lactococcus casei, Lactococcus lactis, and Enterococcus faecium. By testing a panel of L. lactis cell wall mutants, we observed that Lc-Lys and Lc-Lys-2 were not able to lyse mutants with a modified PG cross-bridge, constituting d-Ala⁴→l-Ala-(l-Ala/l-Ser)-l-Lys³; moreover, they do not lyse the L. lactis mutant containing only the nonamidated d-Asp cross-bridge, i.e. d-Ala⁴→d-Asp-l-Lys³. In contrast, Lc-Lys could lyse the ampicillin-resistant E. faecium mutant with 3→3 l-Lys³-d-Asn-l-Lys³ bridges replacing the wild-type 4→3 d-Ala⁴-d-Asn-l-Lys³ bridges. We showed that the C-terminal CWBD of Lc-Lys binds PG containing mainly d-Asn but not PG with only the nonamidated d-Asp-containing cross-bridge, indicating that the CWBD confers to Lc-Lys its narrow specificity. In conclusion, the CWBD characterized in this study is a novel type of PG-binding domain targeting specifically the d-Asn interpeptide bridge of PG.