Identification of the catalytic nucleophile of the family 29 alpha-L-fucosidase from Thermotoga maritima through trapping of a covalent glycosyl-enzyme intermediate and mutagenesis

Fucose-containing glycoconjugates are key antigenic determinants in many biological processes. A change in expression levels of the enzymes responsible for tailoring these glycoconjugates has been associated with many pathological conditions and it is therefore surprising that little information is known regarding the mechanism of action of these important catabolic enzymes. Thermotoga maritima, a thermophilic bacterium, produces a wide range of carbohydrate-processing enzymes including a 52-kDa alpha-L-fucosidase that has 38% sequence identity and 56% similarity to human fucosidases. The catalytic nucleophile of this enzyme was identified to be Asp-224 within the peptide sequence 222WNDMGWPEKGKEDL235 using the mechanism-based covalent inactivator 2-deoxy-2-fluoro-alpha-L-fucosyl fluoride. The 10(4)-fold lower activity (kcat/Km) of the site-directed mutant D224A, and the subsequent rescue of activity upon addition of exogenous nucleophiles, conclusively confirms this assignment. This article presents the first direct identification of the catalytic nucleophile of an alpha-L-fucosidase, a key step in the understanding of these important enzymes.     

Postoligomerization folding of human cytomegalovirus glycoprotein B: identification of folding intermediates and importance of disulfide bonding.

Human cytomegalovirus glycoprotein B (gB or UL55) has been demonstrated to be a disulfide-linked homodimer within the envelope of mature virions. Previously, it has been shown that gB undergoes a rapid dimerization nearly coincident with its synthesis. Following dimerization, the molecule slowly folds into a form which can be transported from the endoplasmic reticulum. In this study we have examined the prolonged folding of gB by using a set of defined gB-reactive murine monoclonal antibodies and gB expressed as a recombinant protein in the absence of other human cytomegalovirus proteins. Our results have documented a folding pathway consistent with the relatively rapid dimerization of the translation product followed by delayed conversion into a fully folded molecule. Assembly of the dominant antigenic domain of gB, AD-1, preceded dimerization and folding of the molecule. The fully folded dimer was heat stable, but its conformation was altered by treatment with 2% sodium dodecyl sulfate (SDS), whereas an oligomeric folding intermediate was both heat and SDS stable. Postoligomerization disulfide bond formation could be demonstrated during folding of gB, suggesting that the formation of these covalent bonds could contribute to the prolonged folding of this glycoprotein.     

On the origin of self-incompatibility haplotypes: transition through self-compatible intermediates

Self-incompatibility (SI) in flowering plants entails the inhibition of fertilization by pollen that express specificities in common with the pistil. In species of the Solanaceae, Rosaceae, and Scrophulariaceae, the inhibiting factor is an extracellular ribonuclease (S-RNase) secreted by stylar tissue. A distinct but as yet unknown gene (provisionally called pollen-S) appears to determine the specific S-RNase from which a pollen tube accepts inhibition. The S-RNase gene and pollen-S segregate with the classically defined S-locus. The origin of a new specificity appears to require, at minimum, mutations in both genes. We explore the conditions under which new specificities may arise from an intermediate state of loss of self-recognition. Our evolutionary analysis of mutations that affect either pistil or pollen specificity indicates that natural selection favors mutations in pollen-S that reduce the set of pistils from which the pollen accepts inhibition and disfavors mutations in the S-RNase gene that cause the nonreciprocal acceptance of pollen specificities. We describe the range of parameters (rate of receipt of self-pollen and relative viability of inbred offspring) that permits the generation of a succession of new specificities. This evolutionary pathway begins with the partial breakdown of SI upon the appearance of a mutation in pollen-S that frees pollen from inhibition by any S-RNase presently in the population and ends with the restoration of SI by a mutation in the S-RNase gene that enables pistils to reject the new pollen type.     

Identification of the catalytic nucleophile in Family 42 beta-galactosidases by intermediate trapping and peptide mapping: YesZ from Bacillus subtilis

The mechanism-based inhibitor 2,4-dinitrophenyl 2-deoxy-2-fluoro-beta-d-galactopyranoside (DNP2FGal) was used to inactivate the Family 42 beta-galactosidase (YesZ) from Bacillus subtilis via the trapping of a glycosyl-enzyme intermediate, thereby tagging the catalytic nucleophile in the active site. Proteolytic digestion of the inactivated enzyme and of a control sample of unlabeled enzyme, followed by comparative high-performance liquid chromatography and mass spectrometric analysis identified a labelled peptide of the sequence ETSPSYAASL. These data, combined with sequence alignments of this region with representative members of Family 42, unequivocally identify the catalytic nucleophile in this enzyme as Glu-295, thereby providing the first direct experimental proof of the identity of this residue within Family 42.     

Point mutation in the human dystrophin gene: identification through western blot analysis

Using antibodies directed against the amino-terminus of dystrophin, we identified a truncated protein in a Duchenne muscular dystrophy patient. Antibodies directed against the carboxy-terminus failed to identify any cross-reactive material, a result consistent with premature termination of dystrophin translation. The estimated molecular mass of 126 kDa predicted the approximate location of the mutation in the mRNA and in the gene. Sequencing of cloned PCR products from patient muscle cDNA revealed a nonsense mutation, which was confirmed by direct sequencing of amplified patient genomic DNA. The mutation, a G to T transversion, at position 3714 changes a glutamic acid codon to an Amber stop codon. Translation of mRNA containing this mutation would be expected to result in a truncated protein with a molecular mass of 133 kDa, in close agreement with the 126 kDa estimated by Western blot analysis. This is the first reported case of a point mutation in this very large human gene.     

Family 18 chitolectins: comparison of MGP40 and HUMGP39

Glycosidase and lectins both bind sugars, but only the glycosidases have catalytic activity. The glycosidases occur among over 100 evolved protein families and Family 18 is one of the two chitinases (EC 3, 2.1.14) families. Interestingly, lectins are also in this evolutionary group of Family 18 glycosidase proteins. The proteins belonging to the enzymatically inactive class are referred to as chitolectins and have a binding site that is highly similar to the catalytic Family 18 enzymes. We present a comparison of the recently obtained structures of two Family 18 chitolectins, MGP40 [A.K. Mohanty, G. Singh, M. Paramasivam, K. Saravanan, T. Jabeen, S. Sharma, S. Yadav, P. Kaur, P. Kumar, A. Srinivasan, T.P. Singh, Crystal structure of a novel regulatory 40kDa mammary gland protein (MGP-40) secreted during involution, J. Biol. Chem. 278 (2003) 14451-14460.] and HumGP39 [F. Fusetti, T. Pijning, K.H. Kalk, E. Bos, B.W. Dijkstra, Crystal structure and carbohydrate-binding properties of the human cartilage glycoprotein-39, J. Biol. Chem. 278 (2003) 37753-37760; D.R. Houston, D.R. Anneliese, C.K. Joanne, D.M.V. Aalten, Structure and ligand-induced conformational change of the 39kDa glycoprotein from human articular chondrocytes, J. Biol. Chem. 278 (2003) 30206-30212.] with a focus on the glycosidase active site. We compare the sequence and the structure of these two Family 18 protein classes. The difference between the active and inactive protein is a glutamic acid which acts as the essential acid/base residue for chitin cleavage and is replaced with leucine or glutamine in the chitolectins. Furthermore, a mechanism for the interaction between the chitolectin and oligosaccharides was proposed.     

The second-shell metal ligands of human arginase affect coordination of the nucleophile and substrate

The active sites of eukaryotic arginase enzymes are strictly conserved, especially the first- and second-shell ligands that coordinate the two divalent metal cations that generate a hydroxide molecule for nucleophilic attack on the guanidinium carbon of l-arginine and the subsequent production of urea and l-ornithine. Here by using comprehensive pairwise saturation mutagenesis of the first- and second-shell metal ligands in human arginase I, we demonstrate that several metal binding ligands are actually quite tolerant to amino acid substitutions. Of >2800 double mutants of first- and second-shell residues analyzed, we found more than 80 unique amino acid substitutions, of which four were in first-shell residues. Remarkably, certain second-shell mutations could modulate the binding of both the nucleophilic water/hydroxide molecule and substrate or product ligands, resulting in activity greater than that of the wild-type enzyme. The data presented here constitute the first comprehensive saturation mutagenesis analysis of a metallohydrolase active site and reveal that the strict conservation of the second-shell metal binding residues in eukaryotic arginases does not reflect kinetic optimization of the enzyme during the course of evolution.     

Human protein aging: modification and crosslinking through dehydroalanine and dehydrobutyrine intermediates

Nonenzymatic post‐translational modification (PTM) of proteins is a fundamental molecular process of aging. The combination of various modifications and their accumulation with age not only affects function, but leads to crosslinking and protein aggregation. In this study, aged human lens proteins were examined using HPLC–tandem mass spectrometry and a blind PTM search strategy. Multiple thioether modifications of Ser and Thr residues by glutathione (GSH) and its metabolites were unambiguously identified. Thirty‐four of 36 sites identified on 15 proteins were found on known phosphorylation sites, supporting a mechanism involving dehydroalanine (DHA) and dehydrobutyrine (DHB) formation through β‐elimination of phosphoric acid from phosphoserine and phosphothreonine with subsequent nucleophilic attack by GSH. In vitro incubations of phosphopeptides demonstrated that this process can occur spontaneously under physiological conditions. Evidence that this mechanism can also lead to protein–protein crosslinks within cells is provided where five crosslinked peptides were detected in a human cataractous lens. Nondisulfide crosslinks were identified for the first time in lens tissue between βB2‐ & βB2‐, βA4‐ & βA3‐, γS‐ & βB1‐, and βA4‐ & βA4‐crystallins and provide detailed structural information on in vivo crystallin complexes. These data suggest that phosphoserine and phosphothreonine residues represent susceptible sites for spontaneous breakdown in long‐lived proteins and that DHA‐ and DHB‐mediated protein crosslinking may be the source of the long‐sought after nondisulfide protein aggregates believed to scatter light in cataractous lenses. Furthermore, this mechanism may be a common aging process that occurs in long‐lived proteins of other tissues leading to protein aggregation diseases.     

Vicinity analysis: a methodology for the identification of similar protein active sites

Vicinity analysis (VA) is a new methodology developed to identify similarities between protein binding sites based on their three-dimensional structure and the chemical similarity of matching residues. The major objective is to enable searching of the Protein Data Bank (PDB) for similar sub-pockets, especially in proteins from different structural and biochemical series. Inspection of the ligands bound in these pockets should allow ligand functionality to be identified, thus suggesting novel monomers for use in library synthesis. VA has been developed initially using the ATP binding site in kinases, an important class of protein targets involved in cell signalling and growth regulation. This paper defines the VA procedure and describes matches to the phosphate binding sub-pocket of cyclin-dependent protein kinase 2 that were found by searching a small test database that has also been used to parameterise the methodology.     

Characterization of antibodies to a rabbit hepatic UDP-glucuronosyltransferase and the identification of an immunologically similar enzyme in human liver

An antibody to a UDP-glucuronosyltransferase (UDPGT) isoenzyme which catalyzes the glucuronidation of p-nitrophenol (PNP) in rabbit liver was raised in sheep and used to identify immunologically similar UDPGTs in rabbit and human livers. Immunoblotting experiments showed that the antisera specifically recognized PNP UDPGT but not estrone UDPGT purified from rabbit liver. Sheep anti-rabbit liver PNP UDPGT IgG immunoprecipitated PNP, 1-naphthol, and 4-methylumbelliferone glucuronidation activities in rabbit and human liver microsomal preparations. In rabbit liver microsomes the antibody did not immunoprecipitate estrone or estradiol glucuronidation activities. In human liver microsomes, 4-aminobiphenyl but not estriol glucuronidation activities were immunoprecipitated, suggesting that the antibody recognizes a specific UDPGT (pI 6.2) in human liver microsomes.     

Folding of dimeric methionine adenosyltransferase III: identification of two folding intermediates

Methionine adenosyl transferase (MAT) is an essential enzyme that synthesizes AdoMet. The liver-specific MAT isoform, MAT III, is a homodimer of a 43.7-kDa subunit that organizes in three nonsequential alpha-beta domains. Although MAT III structure has been recently resolved, little is known about its folding mechanism. Equilibrium unfolding and refolding of MAT III, and the monomeric mutant R265H, have been monitored using different physical parameters. Tryptophanyl fluorescence showed a three-state folding mechanism. The first unfolding step was a folding/association process as indicated by its dependence on protein concentration. The monomeric folding intermediate produced was the predominant species between 1.5 and 3 m urea. It had a relatively compact conformation with tryptophan residues and hydrophobic surfaces occluded from the solvent, although its N-terminal region may be very unstructured. The second unfolding step monitored the denaturation of the intermediate. Refolding of the intermediate showed first order kinetics, indicating the presence of a kinetic intermediate within the folding/association transition. Its presence was confirmed by measuring the 1,8-anilinonaphtalene-8-sulfonic acid binding in the presence of tripolyphosphate. We propose that the folding rate-limiting step is the formation of an intermediate, probably a structured monomer with exposed hydrophobic surfaces, that rapidly associates to form dimeric MAT III.     

Catalytic oxidation of 2,4,5-trihydroxyphenylalanine by tyrosinase: identification and evolution of intermediates.

The oxidation of 3,4-dihydroxyphenylalanine (dopa) by O2 catalyzed by tyrosinase yields 4-(2-carboxy-2-aminoethyl)-1,2-benzoquinone, with its amino group protonated (o-dopaquinone-H+). This evolves non-enzymatically through two branches (cyclization and/or hydroxylation), whose respective operations are determined by pH. The hydroxylation branch of o-dopaquinone-H+ only operates significantly at pH < or = 5.0 and involves the accumulation of 2,4,5-trihydroxyphenylalanine (topa), which has been detected by high-performance liquid chromatography (HPLC). This last compound is also a substrate of tyrosinase. The oxidation of topa by both tyrosinase and periodate yields 5-(2-carboxy-2-aminoethyl)-4-hydroxy-1,2-benzoquinone, with its amino group protonated (o-topaquinone-H+), which is red (RTQH) (lambda max 272-485 nm) at pH 7.0 and yellow (TTQH) (lambda max 265-390 nm) at pH 3.0. This is based on pKa 4.5 of the 2-OH group of the benzene ring of o-topaquinone-H+, as derived from spectrophotometric and HPLC assays. At physiological pH, RTQH undergoes deprotonation of the ammonium group of the side chain to yields RTQ, which cyclize into 2-carboxy-2,3-dihydroxyindolen-5,6-quinone (dopachrome), with a 1:1 stoichiometry and first-order kinetics. The evolution of RTQH has been analyzed by spectrophotometry, HPLC, cyclic voltammetry and constant potential electrolytic assays. From HPLC assays, the value of the first-order constant for the evolution of RTQH at pH 7.0 (kRTQHapp 4.83 x 10(-5) s-1), as well as of the rate constant for the cyclization step of RTQ (kRTQc 2.53 x 10(-3) s-1) were determined.     

Bioactivation of coumarin in rat olfactory mucosal microsomes: Detection of protein covalent binding and identification of reactive intermediates through analysis of glutathione adducts

The presence of high levels, as well as tissue-specific forms, of cytochrome P450 enzymes in mammalian olfactory mucosa (OM) has important implications in the bioactivation and toxicity of xenobiotics entering the tissue. Previous studies have shown that coumarin, a known olfactory toxicant in rats, is bioactivated by OM microsomal P450s to a number of products, presumably via coumarin-3,4-epoxide and other epoxide intermediates. The aim of the current study was to obtain direct evidence for the formation of such reactive intermediates in rat OM through the detection of protein covalent binding and glutathione (GSH) adduct formation. Protein covalent binding experiments with [¹⁴C]coumarin (10 μM) displayed a 7–9-fold higher NADPH-dependent radioactivity binding in rat OM microsomes (2.5 nmol/mg/30 min) compared to those in rat and human liver microsomes; the binding value in rat OM microsomes was substantially but not completely reduced by the addition of GSH (5 mM). LC/MS analyses detected a number of GSH adducts in GSH-supplemented coumarin metabolism reaction in rat OM microsomes; 3-glutathionyl coumarin was found to be the major one, indicating 3,4-epoxidation as the main bioactivation pathway. Additional GSH adducts were identified, presumably forming via the same pathway or epoxidation on the benzene moiety. Our findings provide direct evidence for the formation of multiple coumarin reactive intermediates in rat OM, leading to protein covalent binding and GSH conjugation.     

A bacterial ecto-triphosphate diphosphohydrolase similar to human CD39 is essential for intracellular multiplication of Legionella pneumophila

As part of its pathogenesis, Legionella pneumophila persists within human alveolar macrophages in non-acidified organelles that do not mature into phagolysosomes. Two L. pneumophila genes, lpg0971 and lpg1905, are predicted to encode ecto-nucleoside triphosphate diphosphohydrolases (ecto-NTPDases) that share sequence similarity with human CD39/NTPDase1. The predicted products possess five apyrase conserved domains that are typical of eukaryotic ecto-NTPDases. In this study, we found that an lpg1905 mutant was recovered in lower numbers from macrophages, alveolar epithelial cells and the amoeba, Hartmannella vermiformis compared with wild-type L. pneumophila and an lpg0971 mutant. Similar to human CD39, recombinant purified Lpg1905 exhibited ATPase and ADPase activity and possessed the ability to inhibit platelet aggregation. Mutation of a conserved Glu159 residue that is essential for CD39 activity inhibited ATPase and ADPase activity of Lpg1905. In addition, enzyme activity was inhibited in the presence of the specific ecto-NTPDase inhibitor, ARL67156. The entry and replication defect of the lpg1905 mutant was reversed upon transcomplementation with lpg1905 but not lpg1905E159A encoding an enzymatically inactive form of the protein. Although several protozoan parasites exhibit ecto-NTPDase activity, including Toxoplasma gondii, Trichomonas vaginalis and Trypanosoma cruzi, this is the first time a bacterial ecto-NTPDase has been implicated in virulence.     

The basal and major pilins in the Corynebacterium diphtheriae SpaA pilus adopt similar structures that competitively react with the pilin polymerase

Many species of pathogenic gram-positive bacteria display covalently crosslinked protein polymers (called pili or fimbriae) that mediate microbial adhesion to host tissues. These structures are assembled by pilus-specific sortase enzymes that join the pilin components together via lysine-isopeptide bonds. The archetypal SpaA pilus from Corynebacterium diphtheriae is built by the ^CdSrtA pilus-specific sortase, which crosslinks lysine residues within the SpaA and SpaB pilins to build the shaft and base of the pilus, respectively. Here, we show that ^CdSrtA crosslinks SpaB to SpaA via a K139(SpaB)-T494(SpaA) lysine-isopeptide bond. Despite sharing only limited sequence homology, an NMR structure of SpaB reveals striking similarities with the N-terminal domain of SpaA (^NSpaA) that is also crosslinked by ^CdSrtA. In particular, both pilins contain similarly positioned reactive lysine residues and adjacent disordered AB loops that are predicted to be involved in the recently proposed “latch” mechanism of isopeptide bond formation. Competition experiments using an inactive SpaB variant and additional NMR studies suggest that SpaB terminates SpaA polymerization by outcompeting ^NSpaA for access to a shared thioester enzyme–substrate reaction intermediate.     

Isopentenyldiphosphate:dimethylallyldiphosphate isomerase: construction of a high-level heterologous expression system for the gene from Saccharomyces cerevisiae and identification of an active-site nucleophile

Isopentenyldiphosphate:dimethylallyldiphosphate isomerase (IPP isomerase) is an enzyme in isoprene metabolism which catalyzes the interconversion of the fundamental five-carbon homoallylic and allylic diphosphate building blocks for the pathway. The gene encoding IPP isomerase has recently been isolated from Saccharomyces cerevisiae [Anderson, M. S., Muehlbacher, M., Street, I.P., Proffitt, J., & Poulter, C. D. (1989) J. Biol. Chem. 264, 19169-19175]. A heterologous expression system was constructed for the gene and used to overexpress IPP isomerase in Escherichia coli. In transformants carrying the expression vector, IPP isomerase activity was increased by over 100,000-fold relative to that of the untransformed host strain. The overexpressed enzyme constitutes 30-35% of the total soluble cell protein and can be purified to homogeneity in two steps. Recombinant IPP isomerase was indistinguishable from that purified from yeast. 3-(Fluoromethyl)-3-butenyl diphosphate (FIPP) is a specific active-site-directed inhibitor of IPP isomerase from Claviceps purpurea [Muehlbacher, M., & Poulter, C. D. (1988) Biochemistry 27, 7315-7328]. Inactivation of yeast IPP isomerase by FIPP was active-site-directed, and inhibition resulted in formation of a stoichiometric enzyme-inhibitor complex. The site of covalent attachment in the enzyme-inhibitor complex was determined by inactivating IPP isomerase with [4-3H]FIPP, followed by digestion of the labeled enzyme with trypsin and purification of the resulting radioactive peptides by reversed-phase high-performance liquid chromatography. The primary site of attachment was Cys-139.     

Replication of the Streptomyces plasmid pSN22 through single-stranded intermediates

The replication of the 11 kb conjugative multicopy Streptomyces plasmid pSN22 was analyzed. Mutation and complementation analyses indicated that the minimal region essential for plasmid replication was located on a 1.9 kb fragment of pSN22, containing a trans-acting element encoding a replication protein and a cis-acting sequence acting as a replication origin. Southern hybridization showed that minimal replicon plasmids accumulated much more single-stranded plasmid molecules than did wild-type pSN22. Only one strand was accumulated. A 500 by fragment from the pSN22 transfer region was identified which reduced the relative amount of single-stranded DNA, when added in the native orientation to minimal replicon plasmids. This 500 by DNA sequence may be an origin for second-strand synthesis. It had no effect on the efficiency of co-transformation, plasmid incompatibility, or stability. The results indicate that pSN22 replicates via single-stranded intermediates by a rolling circle mechanism.