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International audience; Flavin coenzymes are universally found in biological redox reactions. DNA photolyases, with their flavin chromophore (FAD), utilize blue light for DNA repair and photoreduction. The latter process involves two single-electron transfers to FAD with an intermittent protonation step to prime the enzyme active for DNA repair. Here we use time-resolved serial femtosecond X-ray crystallography to describe how light-driven electron transfers trigger subsequent nanosecond-to-microsecond entanglement between FAD and its Asn/Arg-Asp redox sensor triad. We found that this key feature within the photolyase-cryptochrome family regulates FAD re-hybridization and protonation. After first electron transfer, the FAD•- isoalloxazine ring twists strongly when the arginine closes in to stabilize the negative charge. Subsequent breakage of the arginine-aspartate salt bridge allows proton transfer from arginine to FAD•-. Our molecular videos demonstrate how the protein environment of redox cofactors organizes multiple electron/proton transfer events in an ordered fashion, which could be applicable to other redox systems such as photosynthesis.

Toll-like receptor 4 (TLR4) recognizes various protein ligands; however, the protein-TLR4 binding model is unclear. Here we demonstrate a Crenomytilus grayanus lectin (CGL)-TLR4/MD2 model to show that CGL interacts with a TLR4/myeloid differentiation factor 2 (MD2) complex independently of sugar-binding properties. CGL could suppress lipopolysaccharide-induced immune responses significantly, suggesting that TLR4 itself has potential as a therapeutic target.

The structure of a Pseudomonas borneol dehydrogenase was determined at 1.84 Å resolution. Major differences from its homologues in the C-terminal helices and the associated loops may suggest determinants for substrate recognition., During the microbial degradation of borneol, a bicyclic plant monoterpene, it is first converted into camphor by borneol dehydrogenase (BDH) and then enters a known camphor-degradation pathway. Previously, a recombinant Pseudomonas BDH was found in inclusion bodies when expressed in Escherichia coli. After refolding, it was still unstable and was difficult to concentrate. Here, the protein-expression conditions were improved by changing the medium from lysogeny broth to Terrific Broth, yielding a soluble form of the enzyme with higher activity. The protein was crystallized and its 3D structure was determined by X-ray diffraction. Like other known homologues such as quinuclidinone reductase, the protein forms a tetramer with subunits containing Rossmann folds. Structural comparison revealed major differences in the C-terminal helices and the associated loops. It is likely that these regions contain the determinants for substrate recognition.

Most known cellulase-associated carbohydrate-binding modules (CBMs) are attached to the N- or C-terminus of the enzyme or are expressed separately and assembled into multi-enzyme complexes (for example to form cellulosomes), rather than being an insertion into the catalytic domain. Here, by solving the crystal structure, it is shown that MtGlu5 from Meiothermus taiwanensis WR-220, a GH5-family endo-β-1,4-glucanase (EC 3.2.1.4), has a bipartite architecture consisting of a Cel5A-like catalytic domain with a (β/α)8 TIM-barrel fold and an inserted CBM29-like noncatalytic domain with a β-jelly-roll fold. Deletion of the CBM significantly reduced the catalytic efficiency of MtGlu5, as determined by isothermal titration calorimetry using inactive mutants of full-length and CBM-deleted MtGlu5 proteins. Conversely, insertion of the CBM from MtGlu5 into TmCel5A from Thermotoga maritima greatly enhanced the substrate affinity of TmCel5A. Bound sugars observed between two tryptophan side chains in the catalytic domains of active full-length and CBM-deleted MtGlu5 suggest an important stacking force. The synergistic action of the catalytic domain and CBM of MtGlu5 in binding to single-chain polysaccharides was visualized by substrate modeling, in which additional surface tryptophan residues were identified in a cross-domain groove. Subsequent site-specific mutagenesis results confirmed the pivotal role of several other tryptophan residues from both domains of MtGlu5 in substrate binding. These findings reveal a way to incorporate a CBM into the catalytic domain of an existing enzyme to make a robust cellulase.

Background K1 capsular polysaccharide (CPS)-associated Klebsiella pneumoniae is the primary cause of pyogenic liver abscesses (PLA) in Asia. Patients with PLA often have serious complications, ultimately leading to a mortality of ~ 5%. This K1 CPS has been reported as a promising target for development of glycoconjugate vaccines against K. pneumoniae infection. The pyruvylation and O-acetylation modifications on the K1 CPS are essential to the immune response induced by the CPS. To date, however, obtaining the fragments of K1 CPS that contain the pyruvylation and O-acetylation for generating glycoconjugate vaccines still remains a challenge. Methods We analyzed the digested CPS products with NMR spectroscopy and mass spectrometry to reveal a bacteriophage-derived polysaccharide depolymerase specific to K1 CPS. The biochemical and biophysical properties of the enzyme were characterized and its crystal structures containing bound CPS products were determined. We also performed site-directed mutagenesis, enzyme kinetic analysis, phage absorption and infectivity studies, and treatment of the K. pneumoniae-infected mice with the wild-type and mutant enzymes. Results We found a bacteriophage-derived polysaccharide lyase that depolymerizes the K1 CPS into fragments of 1–3 repeating trisaccharide units with the retention of the pyruvylation and O-acetylation, and thus the important antigenic determinants of intact K1 CPS. We also determined the 1.46-Å-resolution, product-bound crystal structure of the enzyme, revealing two distinct carbohydrate-binding sites in a trimeric β-helix architecture, which provide the first direct evidence for a second, non-catalytic, carbohydrate-binding site in bacteriophage-derived polysaccharide depolymerases. We demonstrate the tight interaction between the pyruvate moiety of K1 CPS and the enzyme in this second carbohydrate-binding site to be crucial to CPS depolymerization of the enzyme as well as phage absorption and infectivity. We also demonstrate that the enzyme is capable of protecting mice from K1 K. pneumoniae infection, even against a high challenge dose. Conclusions Our results provide insights into how the enzyme recognizes and depolymerizes the K1 CPS, and demonstrate the potential use of the protein not only as a therapeutic agent against K. pneumoniae, but also as a tool to prepare structurally-defined oligosaccharides for the generation of glycoconjugate vaccines against infections caused by this organism.

Description, One-way translocation and processive cleavage of substrate polypeptide occur in each of the Lon proteolytic active sites., The Lon protease is the prototype of a family of proteolytic machines with adenosine triphosphatase modules built into a substrate degradation chamber. Lon is known to degrade protein substrates in a processive fashion, cutting a protein chain processively into small peptides before commencing cleavages of another protein chain. Here, we present structural and biochemical evidence demonstrating that processive substrate degradation occurs at each of the six proteolytic active sites of Lon, which forms a deep groove that partially encloses the substrate polypeptide chain by accommodating only the unprimed residues and permits processive cleavage in the C-to-N direction. We identify a universally conserved acidic residue at the exit side of the binding groove indispensable for the proteolytic activity. This noncatalytic residue likely promotes processive proteolysis by carboxyl-carboxylate interactions with cleaved intermediates. Together, these results uncover a previously unrecognized mechanism for processive substrate degradation by the Lon protease.

Chromoproteins are a good source of engineered biological tools. We previously reported the development of a blue fluorescent protein, termed shBFP, which was derived from a purple chromoprotein shCP found in the sea anemone Stichodacyla haddoni. shBFP contains a Leu

Bacterial polyynes are highly active natural products with a broad-spectrum of antimicrobial activities. However, their detailed mechanism of action remains unclear. Through integrating comparative genomics, transcriptomics, functional genetics, and metabolomics analysis, we identified a unique polyyne resistance gene, masL (encoding acetyl-CoA acetyltransferase), from the biosynthesis gene cluster (BGC) dominant for the production of antifungal polyynes (massilin A, massilin B, collimonin C, and collimonin D) in Massilia sp. YMA4. Phylogenic and chemotaxonomic analyses characterized the core architecture of bacterial polyyne BGC. The crystallographic analysis of the MasL-collimonin C complex indicated that bacterial polyynes serve as a covalent inhibitor of acetyl-CoA acetyltransferase. Moreover, we confirmed that the bacterial polyynes disrupted cell membrane integrity and inhibited cell viability of Candida albicans by targeting ERG10 (homolog of MasL). Overall, understanding of the antifungal mechanism of bacterial polyynes presented herein will be useful for the development of polyynes for fungal infections.

The semi-fossil and pit-less Azemiops feae is possibly the most primitive crotalid species. Here, we have cloned and sequenced cDNAs encoding four serine proteases (vSPs) from the venom glands of Chinese A. feae. Full amino-acid sequences of the major vSP (designated as AzKNa) and three minor vSPs (designated as AzKNb, AzKNc and Az-PA) were deduced. Using Protein-BLAST search, the ten most-similar vSPs for each Azemiops vSP have been selected for multiple sequence alignment, and all the homologs are crotalid vSPs. The results suggest that the A. feae vSPs are structurally most like those of eastern-Chinese Gloydius, Viridovipera, Protobothrops and North American pitvipers, and quite different from more-specialized vSPs such as Agkistrodon venom Protein-C activators. The vSPs from Chinese A. feae and those from Vietnamese A. feae show significant sequence variations. AzKNa is acidic and contains six potential N-glycosylation sites and its surface-charge distribution differs greatly from that of AzKNb, as revealed by 3D-modeling. AzKNb and AzKNc do not contain N-glycosylation sites although most of their close homologs contain one or two. Az-PA belongs to the plasminogen-activator subtype with a conserved N20-glycosylation site. The evolution of this subtype of vSPs in Azemiops and related pitvipers has been traced by phylogenetic analysis.

Proteins with sequence or structure similar to those of di-Zn exopeptidases are usually classified as the M28-family enzymes, including the mammalian-type glutaminyl cyclases (QCs). QC catalyzes protein N-terminal pyroglutamate formation, a posttranslational modification important under many physiological and pathological conditions, and is a drug target for treating neurodegenerative diseases, cancers and inflammatory disorders. Without functional characterization, mammalian QCs and their orthologs remain indistinguishable at the sequence and structure levels from other M28-family proteins, leading to few reported QCs. Here, we show that a low-barrier carboxylic-acid hydrogen-bond network (CAHBN) is required for QC activity and discriminates QCs from M28-family peptidases. We demonstrate that the CAHBN-containing M28 peptidases deposited in the PDB are indeed QCs. Our analyses identify several thousands of QCs from the three domains of life, and we enzymatically and structurally characterize several. For the first time, the interplay between a CAHBN and the binuclear metal-binding center of mammalian QCs is made clear. We found that the presence or absence of CAHBN is a key discriminator for the formation of either the mono-Zn QCs or the di-Zn exopeptidases. Our study helps explain the possible roles of QCs in life.

The vast majority of in vitro structural and functional studies of the activation mechanism of protein kinases use the kinase domain alone. Well-demonstrated effects of regulatory domains or allosteric factors are scarce for serine/threonine kinases. Here we use a site-specifically phosphorylated SCD1-FHA1-kinase three-domain construct of the serine/threonine kinase Rad53 to show the effect of phospho-priming, an in vivo regulatory mechanism, on the autophosphorylation intermediate and specificity. Unphosphorylated Rad53 is a flexible monomer in solution but is captured in an asymmetric enzyme:substrate complex in crystal with the two FHA domains separated from each other. Phospho-priming induces formation of a stable dimer via intermolecular pT-FHA binding in solution. Importantly, autophosphorylation of unprimed and phospho-primed Rad53 produced predominantly inactive pS350-Rad53 and active pT354-Rad53, respectively. The latter mechanism was also demonstrated in vivo. Our results show that, while Rad53 can display active conformations under various conditions, simulation of in vivo regulatory conditions confers functionally relevant autophosphorylation.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe acute respiratory illness with fever, cough and shortness of breath. Up to date, it has resulted in 1826 human infections, including 649 deaths. Analogous to picornavirus 3C protease (3Cpro), 3C-like protease (3CLpro) is critical for initiation of the MERS-CoV replication cycle and is thus regarded as a validated drug target. As presented here, our peptidomimetic inhibitors of enterovirus 3Cpro (6b, 6c and 6d) inhibited 3CLpro of MERS-CoV and severe acute respiratory syndrome coronavirus (SARS-CoV) with IC50 values ranging from 1.7 to 4.7 μM and from 0.2 to 0.7 μM, respectively. In MERS-CoV-infected cells, the inhibitors showed antiviral activity with EC50 values ranging from 0.6 to 1.4 μM, by downregulating the viral protein production in cells as well as reducing secretion of infectious viral particles into culture supernatants. They also suppressed other α- and β-CoVs from human and feline origin. These compounds exhibited good selectivity index (over 70 against MERS-CoV) and could lead to the development of broad-spectrum antiviral drugs against emerging CoVs and picornaviruses.

Summary YbeA from E. coli is a trefoil-knotted SpoU-TrmD (SPOUT) RNA methyltransferase. While its knotted motif plays a key functional role, it is unclear how the knotted topology emerged from evolution. Here, we reverse-engineered an unknotted circular permutant (CP) of YbeA by introducing a new opening at the knotting loop. The resulting CP folded into an unexpected domain-swapped dimer. Untying the knotted loop abrogated its function, perturbed its folding stability and kinetics, and induced allosteric dynamic changes. We speculated that the knotted loop of YbeA is under tension to keep the cofactor in a high-energy configuration while keeping the threading C-terminal helix being knotted. Circular permutation released the mechanical strain thereby allowing the spring-loaded threading helix to flip, to relax, and to form a domain-swapped dimer. Being knotted may be the consequence of selection pressure for the unique structure-function relationship of the SPOUT superfamily that exists in all kingdoms of life.

YbeA from E. coli is a trefoil-knotted SpoU-TrmD (SPOUT) RNA methyltransferase. While its knotted motif plays a key functional role, it is unclear how the knotted topology emerged from evolution. Here, we reverse-engineered an unknotted circular permutant (CP) of YbeA by introducing a new opening at the knotting loop. The resulting CP folded into an unexpected domain-swapped dimer. Untying the knotted loop abrogated its function, perturbed its folding stability and kinetics, and induced allosteric dynamic changes. We speculated that the knotted loop of YbeA is under tension to keep the cofactor in a high-energy configuration while keeping the threading C-terminal helix being knotted. Circular permutation released the mechanical strain thereby allowing the spring-loaded threading helix to flip, to relax and to form a domain-swapped dimer. Being knotted may be the consequence of selection pressure for the unique structure-function relationship of the SPOUT superfamily that exists in all kingdoms of life.

Most plant-produced monoterpenes can be degraded by soil microorganisms. Borneol is a plant terpene that is widely used in traditional Chinese medicine. Neither microbial borneol dehydrogenase (BDH) nor a microbial borneol degradation pathway has been reported previously. One borneol-degrading strain, Pseudomonas sp. strain TCU-HL1, was isolated by our group. Its genome was sequenced and annotated. The genome of TCU-HL1 consists of a 6.2-Mbp circular chromosome and one circular plasmid, pTHL1 (12.6 kbp). Our results suggest that borneol is first converted into camphor by BDH in TCU-HL1 and is further decomposed through a camphor degradation pathway. The recombinant BDH was produced in the form of inclusion bodies. The apparent K m values of refolded recombinant BDH for (+)-borneol and (−)-borneol were 0.20 ± 0.01 and 0.16 ± 0.01 mM, respectively, and the k cat values for (+)-borneol and (−)-borneol were 0.75 ± 0.01 and 0.53 ± 0.01 s −1 , respectively. Two plant BDH genes have been reported previously. The k cat and k cat / K m values of lavender BDH are about 1,800-fold and 500-fold lower, respectively, than those of TCU-HL1 BDH. IMPORTANCE The degradation of borneol in a soil microorganism through a camphor degradation pathway is reported in this study. We also report a microbial borneol dehydrogenase. The k cat and k cat / K m values of lavender BDH are about 1,800-fold and 500-fold lower, respectively, than those of TCU-HL1 BDH. The indigenous borneol- and camphor-degrading strain isolated, Pseudomonas sp. strain TCU-HL1, reminds us of the time 100 years ago when Taiwan was the major producer of natural camphor in the world.

Protein phosphorylation plays an important role in regulating soluble L-glutamic acid decarboxylase (GAD) and membrane-associated GAD activity. Previously, we reported the effect of phosphorylation on the two well-defined GAD isoforms, namely, GAD65 and GAD67, using highly purified preparations of recombinant human brain GAD65 (hGAD65) and GAD67. GAD65 was activated by phosphorylation, while GAD67 was inhibited by phosphorylation. The effect of phosphorylation on GAD65 and GAD67 could be reversed by treatment with protein phosphatases. We further demonstrated that protein kinase A (PKA) and protein kinase C isoform ε were the protein kinases responsible for phosphorylation and regulation of GAD67 and GAD65, respectively. In the current study, using MALDI-TOF, a total of four potential phosphorylation sites were identified in GAD65, two of which (threonine-95 (T-95) and Ser-417) were not reported previously. We have identified one specific phosphorylation site, (T95), in hGAD65 that can be phosphorylated by kinase C ε (PKCε) using MALDITOF. When T95 is mutated to alanine, hGAD65 could no longer be phosphorylated by PKCε, and the effect of PKC-mediated activation on hGAD65 is abolished. However, when T95 is mutated to glutamic acid, which mimics the phosphorylation status of hGAD65, the activity was greatly increased. An increase of GAD65 activity by 55 % compared to the wild type hGAD65 was observed indicating that mutation of T95 to glutamic acid mimics the effect of phosphorylation. A model depicting the role of phosphorylation of GAD65 in regulation of GABA neurotransmission is presented.

Borneol is a bicyclic plant monoterpene. It can be degraded by soil microorganisms through the conversion of borneol dehydrogenase (BDH) and a known camphor degradation pathway. Recombinant BDH from Pseudomonas sp. TCU-HL1 was produced in the form of inclusion body. The refolded BDH1 tends to precipitate. Insoluble recombinant BDH1 was converted into a soluble form by adding glycerol in LB medium. The kcat and kcat/Km values of soluble form BDH1 for (+)-borneol turned out to be about 34-fold and 45-fold higher, respectively, than those of the refolded enzyme. On the other hand, a gene knockout mutant, TCU-HL1Δbdh, was constructed to investigate the possible presence of a second copy of the bdh gene in TCU-HL1 genome. A new gene, bdh2, encoding a BDH isozyme, was identified, and the recombinant BDH2 protein was produced in a soluble form. Both bdh1 and bdh2 genes are expressed in the crude extract of wild type TCU-HL1, as shown by RT-qPCR results. Both BDH isozymes prefer to degrade (+)-borneol, rather than (-)-borneol, probably because (+)-camphor is the main form present in nature.

Most plant-produced monoterpenes can be degraded by soil microorganisms. Borneol is a plant terpene that is widely used in traditional Chinese medicine. Neither microbial borneol dehydrogenase (BDH) nor a microbial borneol degradation pathway has been reported previously. One borneol-degrading strain, Pseudomonas sp. strain TCU-HL1, was isolated by our group. Its genome was sequenced and annotated. The genome of TCU-HL1 consists of a 6.2-Mbp circular chromosome and one circular plasmid, pTHL1 (12.6 kbp). Our results suggest that borneol is first converted into camphor by BDH in TCU-HL1 and is further decomposed through a camphor degradation pathway. The recombinant BDH was produced in the form of inclusion bodies. The apparent KThe degradation of borneol in a soil microorganism through a camphor degradation pathway is reported in this study. We also report a microbial borneol dehydrogenase. The k

Forkhead-associated (FHA) domain is the only signaling domain that recognizes phosphothreonine (pThr) specifically. TRAF-interacting protein with an FHA domain (TIFA) was shown to be involved in immune responses by binding with TRAF2 and TRAF6. We recently reported that TIFA is a dimer in solution and that, upon stimulation by TNF-α, TIFA is phosphorylated at Thr9, which triggers TIFA oligomerization via pThr9-FHA domain binding and activates nuclear factor κB (NF-κB). However, the structural mechanism for the functionally important TIFA oligomerization remains to be established. While FHA domain-pThr binding is known to mediate protein dimerization, its role in oligomerization has not been demonstrated at the structural level. Here we report the crystal structures of TIFA (residues 1-150, with the unstructured C-terminal tail truncated) and its complex with the N-terminal pThr9 peptide (residues 1-15), which show unique features in the FHA structure (intrinsic dimer and extra β-strand) and in its interaction with the pThr peptide (with residues preceding rather than following pThr). These structural features support previous and additional functional analyses. Furthermore, the structure of the complex suggests that the pThr9-FHA domain interaction can occur only between different sets of dimers rather than between the two protomers within a dimer, providing the structural mechanism for TIFA oligomerization. Our results uncover the mechanism of FHA domain-mediated oligomerization in a key step of immune responses and expand the paradigm of FHA domain structure and function.

Acutobin, the α-fibrinogenase from Deinagkistrodon acutus venom, contains four N-glycosylation sites with disialylated complex-typed glycans. Here, we explore the functional roles of each of the N-glycan by site-directed mutagenesis. The wild-type (ATB-wt) and single glycan-knockout mutants of recombinant acutobin were prepared from HEK293T, demonstrating that mutations at Asn(77), Asn(81) and Asn(100) impaired the folding while the S79A mutant and various Asn(229)-deglycosylated mutants were correctly folded. Based on homology modeling of acutobin and multiple sequence alignment with various venom thrombin-like enzymes, the importance of a hydrophilic environment at each glycosylation site to the enzyme folding could be rationalized. Remarkably, all the mutants showed similar catalytic activities for the chromogenic substrate and similar thermal stabilities as ATB-wt, suggesting that the glycan knockout did not affect the gross conformation and stability of the active sites. Although SDS-PAGE analyses revealed that ATB-wt and the D229-mutant degraded all human fibrinogen subunits faster but less specifically in vitro as compared with other mutants that cleaved only the α-subunit, ATB-wt and D229-mutant were not able to release fibrinogen-peptide A and thus coagulated human plasma slower than the other mutants did. In the mice model, the defibrinogenating effect of ATB-wt was stronger and lasting-longer than those of all the mutants. Taken together, all the glycans contribute to the pharmacokinetics of acutobin and ATB-wt in vivo, and the microenvironment around the Asn(229)-glycan appears to regulate the fibrinogen-chain specificity of acutobin while the N-glycans at positions 77, 81 and 100 are crucial for its folding.

Discovery of new antibiotics for combating methicillin-resistant Staphylococcus aureus (MRSA) is of vital importance in the post-antibiotic era. Here, we report four avenaciolide derivatives (1-4) isolated from Neosartorya fischeri, three of which had significant antimicrobial activity against MRSA. The morphology of avenaciolide-treated cells was protoplast-like, which indicated that cell wall biosynthesis was interrupted. Comparing the structures and minimum inhibitory concentrations of 1-4, the α,β-unsaturated carbonyl group seems to be an indispensable moiety for antimicrobial activity. Based on a structural similarity survey of other inhibitors with the same moiety, we revealed that MurA was the drug target. This conclusion was validated by (31)P NMR spectroscopy and MS/MS analysis. Although fosfomycin, which is the only clinically used MurA-targeted antibiotic, is ineffective for treating bacteria harboring the catalytically important Cys-to-Asp mutation, avenaciolides 1 and 2 inhibited not only wild-type but also fosfomycin-resistant MurA in an unprecedented way. Molecular simulation revealed that 2 competitively perturbs the formation of the tetrahedral intermediate in MurA. Our findings demonstrated that 2 is a potent inhibitor of MRSA and fosfomycin-resistant MurA, laying the foundation for the development of new scaffolds for MurA-targeted antibiotics.

Dehydroascorbate reductase (DHAR) is a key enzyme for glutathione (GSH)-dependent reduction of dehydroascorbate (DHA) to recycled ascorbate (AsA) in plants, and plays a major role against the toxicity of reactive oxygen species (ROS). Previously, we proposed that the increase of AsA regeneration via enhanced DHAR activity modulates the ascorbate-glutathione cycle activity against photooxidative stress in Chlamydomonas reinhardtii. In the present work, we use site-directed mutagenesis and crystal structure analysis to elucidate the molecular basis of how C. reinhardtii DHAR (CrDHAR1) is involved in the detoxification mechanisms. Mutagenesis data show that the D21A, D21N and C22A mutations result in severe loss of the enzyme's function, suggesting crucial roles of Asp-21 and Cys-22 in substrate binding and catalysis. The mutant K11A also exhibits reduced redox activity (∼50%). The crystal structure of apo CrDHAR1 further provides insights into the proposed mechanism centering on the strictly conserved Cys-22, which is suggested to initiate the redox reactions of DHA and GSH. Furthermore, in vitro oxidation of the recombinant CrDHAR1 in the presence of 1 mM H2O2 has minor effects on the Km for the substrates but significantly reduces the kcat. The enzyme's activity and its mRNA abundance in the C. reinhardtii cells are increased by treatment with 0.2-1 mM H2O2 but decreased when H2O2 is ≥ 1.5 mM. The latter decrease is accompanied by oxidative damage and lower AsA concentrations. These biochemical and physiological data provide new insights into the catalytic mechanism of CrDHAR1, which protects the C. reinhardtii cells from oxidative stress-induced toxicity.

With an increase in antibiotic-resistant strains, the nosocomial pathogen Acinetobacter baumannii has become a serious threat to global health. Glycoconjugate vaccines containing fragments of bacterial exopolysaccharide (EPS) are an emerging therapeutic to combat bacterial infection. Herein, we characterize the bacteriophage ΦAB6 tailspike protein (TSP), which specifically hydrolyzed the EPS of A. baumannii strain 54149 (Ab-54149). Ab-54149 EPS exhibited the same chemical structure as two antibiotic-resistant A. baumannii strains. The ΦAB6 TSP-digested products comprised oligosaccharides of two repeat units, typically with stoichiometric pseudaminic acid (Pse). The 1.48-1.89-Å resolution crystal structures of an N-terminally-truncated ΦAB6 TSP and its complexes with the semi-hydrolyzed products revealed a trimeric β-helix architecture that bears intersubunit carbohydrate-binding grooves, with some features unusual to the TSP family. The structures suggest that Pse in the substrate is an important recognition site for ΦAB6 TSP. A region in the carbohydrate-binding groove is identified as the determinant of product specificity. The structures also elucidated a retaining mechanism, for which the catalytic residues were verified by site-directed mutagenesis. Our findings provide a structural basis for engineering the enzyme to produce desired oligosaccharides, which is useful for the development of glycoconjugate vaccines against A. baumannii infection.

The Lon proteases are a unique family of chambered proteases with a built-in AAA+ (ATPases associated with diverse cellular activities) module. Here, crystal structures of a unique member of the Lon family with no intrinsic ATPase activity in the proteolytically active form are reported both alone and in complexes with three covalent inhibitors: two peptidomimetics and one derived from a natural product. This work reveals the unique architectural features of an ATP-independent Lon that selectively degrades unfolded protein substrates. Importantly, these results provide mechanistic insights into the recognition of inhibitors and polypeptide substrates within the conserved proteolytic chamber, which may aid the development of specific Lon-protease inhibitors.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe acute respiratory illness with fever, cough and shortness of breath. Up to date, it has resulted in 1826 human infections, including 649 deaths. Analogous to picornavirus 3C protease (3Cpro), 3C-like protease (3CLpro) is critical for initiation of the MERS-CoV replication cycle and is thus regarded as a validated drug target. As presented here, our peptidomimetic inhibitors of enterovirus 3Cpro (6b, 6c and 6d) inhibited 3CLpro of MERS-CoV and severe acute respiratory syndrome coronavirus (SARS-CoV) with IC50 values ranging from 1.7 to 4.7 μM and from 0.2 to 0.7 μM, respectively. In MERS-CoV-infected cells, the inhibitors showed antiviral activity with EC50 values ranging from 0.6 to 1.4 μM, by downregulating the viral protein production in cells as well as reducing secretion of infectious viral particles into culture supernatants. They also suppressed other α- and β-CoVs from human and feline origin. These compounds exhibited good selectivity index (over 70 against MERS-CoV) and could lead to the development of broad-spectrum antiviral drugs against emerging CoVs and picornaviruses., Graphical abstract Image 1, Highlights • Aldehyde-containing peptidomimetics were identified to be potent inhibitors against MERS-CoV 3CLpro. • The active inhibitor showed sub-μM EC50 in killing MERS-CoV. • Compounds were also effective against other α and β-CoVs of both human and feline origin. • We identified broad-spectrum antiviral agents effective against both coronaviruses and picornaviruses.

Immunotoxins are an important class of antibody-based therapeutics. The potency of the immunotoxins depends on the antibody fragments as the guiding modules targeting designated molecules on cell surfaces. Phage-displayed synthetic antibody scFv libraries provide abundant antibody fragment candidates as targeting modules for the immunoconjugates, but the discovery of optimally functional immunoconjugates is limited by the scFv-payload conjugation procedure. In this work, cytotoxicity screening of non-covalently assembled immunotoxins was developed in high throughput format to discover highly functional synthetic antibody fragments for delivering toxin payloads. The principles governing the efficiency of the antibodies as targeting modules have been elucidated from large volume of cytotoxicity data: (a) epitope and paratope of the antibody-based targeting module are major determinants for the potency of the immunotoxins; (b) immunotoxins with bivalent antibody-based targeting modules are generally superior in cytotoxic potency to those with corresponding monovalent targeting module; and (c) the potency of the immunotoxins is positively correlated with the densities of the cell surface antigen. These findings suggest that screening against the target cells with a large pool of antibodies from synthetic antibody libraries without the limitations of natural antibody responses can lead to optimal potency and minimal off-target toxicity of the immunoconjugates.

In this study, we report the structure and function of a lectin from the sea mollusk Crenomytilus grayanus collected from the sublittoral zone of Peter the Great Bay of the Sea of Japan. The crystal structure of C. grayanus lectin (CGL) was solved to a resolution of 1.08 Å, revealing a β-trefoil fold that dimerizes into a dumbbell-shaped quaternary structure. Analysis of the crystal CGL structures bound to galactose, galactosamine, and globotriose Gb3 indicated that each CGL can bind three ligands through a carbohydrate-binding motif involving an extensive histidine- and water-mediated hydrogen bond network. CGL binding to Gb3 is further enhanced by additional side-chain-mediated hydrogen bonds in each of the three ligand-binding sites. NMR titrations revealed that the three binding sites have distinct microscopic affinities toward galactose and galactosamine. Cell viability assays showed that CGL recognizes Gb3 on the surface of breast cancer cells, leading to cell death. Our findings suggest the use of this lectin in cancer diagnosis and treatment.

QC (glutaminyl cyclase) catalyses the formation of N-terminal pGlu (pyroglutamate) in peptides and proteins. pGlu formation in chemoattractants may participate in the regulation of macrophage activation and migration. However, a clear molecular mechanism for the regulation is lacking. The present study examines the role of QC-mediated pGlu formation on MCPs (monocyte chemoattractant proteins) in inflammation. We demonstrated in vitro the pGlu formation on MCPs by QC using MS. A potent QC inhibitor, PBD150, significantly reduced the N-terminal uncyclized-MCP-stimulated monocyte migration, whereas pGlu-containing MCP-induced cell migration was unaffected. QC small interfering RNA revealed a similar inhibitory effect. Lastly, we demonstrated that inhibiting QC can attenuate cell migration by lipopolysaccharide. These results strongly suggest that QC-catalysed N-terminal pGlu formation of MCPs is required for monocyte migration and provide new insights into the role of QC in the inflammation process. Our results also suggest that QC could be a drug target for some inflammatory disorders.

Aberrant pyroglutamate formation at the N terminus of certain peptides and proteins, catalyzed by glutaminyl cyclases (QCs), is linked to some pathological conditions, such as Alzheimer disease. Recently, a glutaminyl cyclase (QC) inhibitor, PBD150, was shown to be able to reduce the deposition of pyroglutamate-modified amyloid-β peptides in brain of transgenic mouse models of Alzheimer disease, leading to a significant improvement of learning and memory in those transgenic animals. Here, we report the 1.05-1.40 Å resolution structures, solved by the sulfur single-wavelength anomalous dispersion phasing method, of the Golgi-luminal catalytic domain of the recently identified Golgi-resident QC (gQC) and its complex with PBD150. We also describe the high-resolution structures of secretory QC (sQC)-PBD150 complex and two other gQC-inhibitor complexes. gQC structure has a scaffold similar to that of sQC but with a relatively wider and negatively charged active site, suggesting a distinct substrate specificity from sQC. Upon binding to PBD150, a large loop movement in gQC allows the inhibitor to be tightly held in its active site primarily by hydrophobic interactions. Further comparisons of the inhibitor-bound structures revealed distinct interactions of the inhibitors with gQC and sQC, which are consistent with the results from our inhibitor assays reported here. Because gQC and sQC may play different biological roles in vivo, the different inhibitor binding modes allow the design of specific inhibitors toward gQC and sQC.

Glutaminyl cyclases (QCs) (EC 2.3.2.5) catalyze the formation of pyroglutamate (pGlu) at the N-terminus of many proteins and peptides, a critical step for the maturation of these bioactive molecules. Proteins having QC activity have been identified in animals and plants, but not in bacteria. Here, we report the first bacterial QC from the plant pathogen Xanthomonas campestris (Xc). The crystal structure of the enzyme was solved and refined to 1.44-A resolution. The structure shows a five-bladed beta-propeller and exhibits a scaffold similar to that of papaya QC (pQC), but with some sequence deletions and conformational changes. In contrast to the pQC structure, the active site of XcQC has a wider substrate-binding pocket, but its accessibility is modulated by a protruding loop acting as a flap. Enzyme activity analyses showed that the wild-type XcQC possesses only 3% QC activity compared to that of pQC. Superposition of those two structures revealed that an active-site glutamine residue in pQC is substituted by a glutamate (Glu(45)) in XcQC, although position 45 is a glutamine in most bacterial QC sequences. The E45Q mutation increased the QC activity by an order of magnitude, but the mutation E45A led to a drop in the enzyme activity, indicating the critical catalytic role of this residue. Further mutagenesis studies support the catalytic role of Glu(89) as proposed previously and confirm the importance of several conserved amino acids around the substrate-binding pocket. XcQC was shown to be weakly resistant to guanidine hydrochloride, extreme pH, and heat denaturations, in contrast to the extremely high stability of pQC, despite their similar scaffold. On the basis of structure comparison, the low stability of XcQC may be attributed to the absence of both a disulfide linkage and some hydrogen bonds in the closure of beta-propeller structure. These results significantly improve our understanding of the catalytic mechanism and extreme stability of type I QCs, which will be useful in further applications of QC enzymes.

The crystal structure of XC1258 from Xanthomonas campestris: A putative procaryotic Nit protein with an arsenic adduct in the active site Ko-Hsin Chin, Ying-Der Tsai, Nei-Li Chan, Kai-Fa Huang, Andrew H.-J. Wang, and Shan-Ho Chou* 1 National Chung Hsing University Biotechnology Center, National Chung-Hsing University, Taichung, 40227, Taiwan, Republic of China 2 Institute of Biochemistry, National Chung-Hsing University, Taichung, 40227, Taiwan, Republic of China 3 Core Facility for Protein Crystallography, Academia Sinica, Nankang, Taipei, Taiwan, Republic of China 4 Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei, Taiwan, Republic of China

The Azemiops snakes are pit-less and phylogenetically located at the Crotalinae and Viperinae divergence. cDNAs encoding five Azemiops venom phospholipase (sPLA2) molecules were cloned and sequenced; their signal-peptides were similar to those of crotalid sPLA2s. Based on their calculated pI-values and residue-49 substitutions, they were designated as Af-E6, Af-N49a, Af-N49a1, Af-N49a2, and Af-N49b, respectively. The first three isoforms, comprising 3-4% of the venom proteins, were purified by reversed-phase HPLC. Af-E6 is catalytically active and has80% sequence-similarity to other Glu(6)-PLA2 (a pitviper venom-marker). Results of phylogenetic analyses reveal that acidic Af-N49a and Af-N49a1 are rather unique and loosely linked with crotalid PLA2s, while Af-N49b is related to the viperid PLA2s with Ser(1) substitution. Notably, the Asn(49)-substitutions in these molecules imply catalytic-independent mechanisms. The 3D-models of Af-E6 and Af-N49a have surface electropotential maps similar to each other and to those of antiplatelet PLA2s, while the Af-N49b model is similar to basic and myotoxic sPLA2 molecules. From Azemiops feae and four other Viperidae, we cloned five novel Cys-rich secretory proteins (CRISPs). Azemiops CRISP and natriuretic-peptide precursors share more sequence similarities with those of crotalid venoms than with viperid venoms, further supporting the theory that Azemiops are sister taxons to pit vipers, especially Tropedolaemus.

N-terminal pyroglutamate (pGlu) formation from its glutaminyl (or glutamyl) precursor is required in the maturation of numerous bioactive peptides. The aberrant formation of pGlu may be related to several pathological processes, such as osteoporosis and amyloidotic diseases. This N-terminal cyclization reaction, once thought to proceed spontaneously, is greatly facilitated by the enzyme glutaminyl cyclase (QC). To probe this important but poorly understood modification, we present here the structure of human QC in free form and bound to a substrate and three imidazole-derived inhibitors. The structure reveals an α/β scaffold akin to that of two-zinc exopeptidases but with several insertions and deletions, particularly in the active-site region. The relatively closed active site displays alternate conformations due to the different indole orientations of Trp-207, resulting in two substrate (glutamine t -butyl ester)-binding modes. The single zinc ion in the active site is coordinated to three conserved residues and one water molecule, which is replaced by an imidazole nitrogen upon binding of the inhibitors. Together with structural and kinetic analyses of several active-site-mutant enzymes, a catalysis mechanism of the formation of protein N-terminal pGlu is proposed. Our results provide a structural basis for the rational design of inhibitors against QC-associated disorders.

Platelet glycoprotein Ib (GPIb)-binding proteins (GPIb-BPs) from snake venoms are usually C-type lectins, which target specific sites of GPIbalpha and elicit distinct effects on platelets. In the present paper, we report a tetrameric platelet-agglutinating factor (molecular mass 121.1 kDa), termed mucrocetin, purified from the venom of Taiwan habu (Trimeresurus mucrosquamatus ). Mucrocetin is a GPIbalpha agonist with a binding site distinct from that of flavocetin-A (a snake venom GPIbalpha antagonist) on GPIbalpha, in spite of the high sequence identity (94.6%) between the two venom lectins. The crystal structure of mucrocetin was solved and refined to 2.8 A (1 A=0.1 nm) resolution, which shows an interesting crystal packing of six-layer cylinders of doughnut-shaped molecules. The four alphabeta heterodimers are arranged in an unusual square-shaped ring stabilized by four interdimer 'head-to-tail' disulphide bridges. Detailed structural comparison between mucrocetin and flavocetin-A suggests that their disparate platelet effects are probably attributable to different charge distributions on the putative concave binding surface. A unique positively charged patch on the binding surface of mucrocetin, formed by Lys102, Lys108, Lys109 and Arg123 in the alpha-subunit coupled with Lys22, Lys102, Lys116 and Arg117 in the beta-subunit, appears to be the primary determinant of its platelet-agglutinating activity. Conceivably, this interesting venom factor may provide a useful tool to study platelet agglutination by binding to the GPIb-IX-V complex.

Mammalian MDC1 interacts with CHK2 in the regulation of DNA damage-induced S-phase checkpoint and apoptosis, which is directed by the association of MDC1-FHA and CHK2-pThr68. However, different ligand specificities of MDC1-FHA have been reported, and no structure is available. Here we report the crystal structures of MDC1-FHA and its complex with a CHK2 peptide containing pThr68. Unlike other FHA domains, MDC1-FHA exists as an intrinsic dimer in solution and in crystals. Structural and binding analyses support pThr+3 ligand specificity and provide structural insight into MDC1-CHK2 interaction.

Venoms from crotalid and viperid snakes contain several peptide inhibitors which regulate the proteolytic activities of their snake-venom metalloproteinases (SVMPs) in a reversible manner under physiological conditions. In this report, we describe the high-resolution crystal structures of a SVMP, TM-3, from Taiwan habu (Trimeresurus mucrosquamatus) cocrystallized with the endogenous inhibitors pyroGlu-Asn-Trp (pENW), pyroGlu-Gln-Trp (pEQW) or pyroGlu-Lys-Trp (pEKW). The binding of inhibitors causes some of the residues around the inhibitor-binding environment of TM-3 to slightly move away from the active-site center, and displaces two metal-coordinated water molecules by the C-terminal carboxylic group of the inhibitors. This binding adopts a retro-manner principally stabilized by four possible hydrogen bonds. The Trp indole ring of the inhibitors is stacked against the imidazole of His143 in the S-1 site of the proteinase. Results from the study of synthetic inhibitor analogues showed the primary specificity of Trp residue of the inhibitors at the P-1 site, corroborating the stacking effect observed in our structures. Furthermore, we have made a detailed comparison of our structures with the binding modes of other inhibitors including batimastat, a hydroxamate inhibitor, and a barbiturate derivative. It suggests a close correlation between the inhibitory activity of an inhibitor and its ability to fill the S-1 pocket of the proteinase. Our work may provide insights into the rational design of small molecules that bind to this class of zinc-metalloproteinases.

This study synthesized a series of hydroxyl-functionalized 2-arylbenzo[b]furans based on the structure of tournefolic acid A and evaluated them for antioxidant and α-glucosidase inhibitory activities. Compounds 5a, 5e, and 5n showed remarkable inhibition of α-glucosidase (IC50 values of 1.9–3.0 μM), and they appear to be even more potent than quercetin. A kinetic binding study indicated that compounds 5a and 5n used a mechanism of mixed-competition to inhibit α-glucosidase. This study also revealed that compounds 5a and 5n bind to either the α-glucosidase or α-glucosidase-4-NPGP complex. Using the crystal structure of the Saccharomyces cerevisiae α-glucosidase, the molecular docking study has predicted the binding of compounds 5a and 5n to the active site of α-glucosidase through both hydrophobic and hydrogen interactions. A DPPH radical scavenging assay further showed that most hydroxyl-functionalized 2-arylbenzo[b]furans possess antioxidant activity. The exception was compound 5p, which has only one hydroxyl group on the 2-phenyl ring of 2-arylbenzo[b]furan. Our results indicate that hydroxyl-functionalized 2-arylbenzo[b]furans possess both antidiabetic as well as antioxidant properties.

Carnosine (CAR) is an endogenous peptide and present in lens, but there is little evidence for its effectiveness in calpain-induced proteolysis inhibition and its differential effects toward different wavelengths of ultraviolet (UV) irradiation. This study aimed to develop three in vitro cataract models to compare the mechanisms underlying the protective activities of CAR. Crude crystallins extracted from porcine lenses were used for antiproteolysis assays, and purified γ-crystallins were used for anti-UV assays. The turbidity in those in vitro models mimics cataract formation and was assayed by measuring optical density (OD) at 405 nm. The effectiveness of CAR on calpain-induced proteolysis was studied at 37 and 58 °C. Patterns of proteins were then analyzed by SDS-PAGE. The turbidity was reduced significantly (p0.05) at 60 min measurements with the increased concentration of CAR (10-300 mM). SDS-PAGE showed that the decreased intensities at both ∼28 and ∼30 kDa protein bands in heat-enhanced assays were ameliorated by CAR at ≥10 mM concentrations. In UV-B studies, CAR (200, 300 mM) reduced the turbidity of γ-crystallin significantly (p0.05) at 6 h observations. The turbidity of samples containing γ-crystallins was ameliorated while incubated with CAR (100, 300 mM) significantly (p0.05) following 4 h of exposure to UV-C. SDS-PAGE showed that the presence of CAR reduced UV-B-induced aggregation of γ-crystallins at ∼44 kDa and resulted in less loss of γ-crystallin following UV-C exposure. The result of modeling also suggests that CAR acts as an inhibitor of calpain. In conclusion, CAR protects lens proteins more readily by inhibiting proteolysis and UV-C-induced degradation than aggregation induced by UV-B irradiation.

Three small peptide components were isolated and purified from the venom of Taiwan habu ( Trimeresurus mucrosquamatus ), which show specific activity to inhibit the strong proteolytic activity of multiple metalloproteinases present in the crude venom. Using multiple chromatographies coupled with successive ultrafiltrations, three inhibitors, i.e. pyroglutamate-lysine-tryptophan (pyroGlu-Lys-Trp), pyroglutamate-asparagine-tryptophan (pyroGlu-Asn-Trp) and pyroglutamate-glutamine-tryptophan (pyroGlu-Gln-Trp) were obtained in good yields and high homogeneity. The yields of these peptide fractions were estimated to be about 0.65 mg, 0.55 mg and 0.42 mg from 250 mg total lyophilized crude venom, which corresponded to the approximate concentrations of 8.4 mM, 7.3 mM and 5.4 mM respectively in venom secretion. Detailed and unambiguous structural determination was established by amino acid analyses, mass spectrometry and microsequencing of purified peptides. Further functional characterization of these three tripeptides showed that they could weakly inhibit three metalloproteinases previously isolated from the same venom. The inhibitory activities were similar among these tripeptides and their IC 50 (concentration for 50% inhibition) were estimated in a range of 0.20-0.95 mM, which is much more effective than citrate, another venom protease inhibitor of low molecular-weight component. Since these tripeptides are the endogenous peptide inhibitors present in the lumen of venom glands, it is conceivable that they may act as a self-defensive mechanism against the auto-digestive deleterious effect of the strong metalloproteinases in vivo, particularly several zinc-dependent metalloproteinases present in crotalid and viperid venoms.

The Lon protease is ubiquitous in nature. Its proteolytic activity is associated with diverse cellular functions ranging from maintaining proteostasis under normal and stress conditions to regulating cell metabolism. Although Lon was originally identified as an ATP-dependent protease with fused AAA+ (ATPases associated with diverse cellular activities) and protease domains, analyses have recently identified LonC as a class of Lon-like proteases with no intrinsic ATPase activity. In contrast to the canonical ATP-dependent Lon present in eukaryotic organelles and prokaryotes, LonC contains an AAA-like domain that lacks the conserved ATPase motifs. Moreover, the LonC AAA-like domain is inserted with a large domain predicted to be largely α-helical; intriguingly, this unique Lon-insertion domain (LID) was disordered in the recently determined full-length crystal structure of Meiothermus taiwanensis LonC (MtaLonC). Here, the crystal structure of the N-terminal AAA-like α/β subdomain of MtaLonC containing an intact LID, which forms a large α-helical hairpin protruding from the AAA-like domain, is reported. The structure of the LID is remarkably similar to the tentacle-like prong of the periplasmic chaperone Skp. It is shown that the LID of LonC is involved both in Skp-like chaperone activity and in recognition of unfolded protein substrates. The structure allows the construction of a complete model of LonC with six helical hairpin extensions defining a basket-like structure atop the AAA ring and encircling the entry portal to the barrel-like degradation chamber of Lon.

The crystal structure of TM-1, a P-I class snake-venom metalloproteinase (SVMP) from the Trimeresurus mucrosquamatus venom, was determined at 1.8-A resolution. The structure exhibits the typical feature of SVMPs and is stabilized by three disulfide linkages. The active site shows a deep S1' substrate-binding pocket limited by the non-conserved Pro174 at the bottom. Further comparisons with other SVMPs suggest that the deep S1' site of TM-1 correlates with its high inhibition sensitivity to the endogenous tripeptide inhibitors. Proteolytic specificity analysis revealed that TM-1 prefers substrates having a moderate-size and hydrophobic residue at the P1' position, consistent with our structural observation.

One novel venom factor was isolated and purified from the venom of Taiwan habu (Trimeresurus mucrosquamatus) using two consecutive anion-exchange and gel-filtration chromatographies followed by cation-exchange HPLC. Further characterization of the purified protein indicated that it lacks the proteolytic activity toward fibrinogen molecules, suggesting that this protein factor does not belong to the familes of metalloproteinases and thrombin-like serine proteases commonly found in the crude venoms of various crotalid snakes. The purified protein exists as a native dimeric protein of 26 kDa, consisting of two closely similar subunits of 16 and 13 kDa, held together by disulfide linkage. N-Terminal sequence analysis revealed that both chains are homologous to each other at the N-terminal fragment and also similar to the factors IX/X-binding protein isolated from Trimeresurus flavoviridis and botrocetin from Bothrops jararaca. This study points to the existence of one new two-chain venom factor without fibrinogenase activity from Taiwan habu, which, in contrast to botrocetin, promotes platelet agglutination even in the absence of von Willebrand factor. Unlike factors IX/X-binding proteins, it did not show affinity to coagulation factors IX and X in the presence of Ca2+ ion. It also shows no inhibition on thrombin, in contrast with bothrojaracin, a thrombin inhibitor isolated from Bothrops jararaca venom. We have therefore named this novel venom factor trimecetin to distinguish it from some structurally related venom factors present in various crotalid and viperid snakes.

Three fibrinogenolytic proteases were isolated and purified from the venom of Taiwan habu (Trimeresurus mucrosquamatus) using anion-exchange and gel-filtration chromatographies followed by cation-exchange HPLC. Further characterization of these purified fractions with fibrinogenase activity indicated that they are single-chain proteases of approximately 24 kDa, possessing strong cleaving activity mainly on the A alpha and less on B beta and gamma chains of fibrinogen subunit chains. Enzyme activities were strongly inhibited by EDTA or 1,10-phenanthroline and not by phenylmethanesulfonyl fluoride, suggesting that these fibrinogenases belong to the family of metalloproteinases and not thrombin-like serine proteases. N-Terminal sequence analysis of these proteases failed to show any free amino-terminal residues, thus hampering the sequence determination by conventional sequencing strategy. Microsequencing on the electroblotted fragments of CNBr-treated proteases separated on SDS-PAGE was then used to determine the partial sequences. Sequence comparison of the determined partial sequences of these proteins with published sequences of the protein data bank revealed that they showed sequence homology with H2-protease. HR2a and protrigramin, which were all shown to belong to metalloproteinases present in various snake venoms. Polymerase chain reaction (PCR) was employed to amplify cDNAs constructed from the poly(A)+RNA of fresh venom glands of the same snake species to facilitate cloning and sequencing of these proteases. Sequencing several positive clones containing amplified cDNAs revealed the existence of one fibrinogenase in the Taiwan habu, which was contained within one complete cDNA encoding the preproproteinase precursor of hemorrhagic metalloproteinases.