Overproduction and one-step purification of the N 5,N 10-methenyltetrahydro-methanopterin cyclohydrolase (Mch) from the hyperthermophilic Methanopyrus kandleri

N ⁵,N ¹⁰-Methenyltetrahydromethanopterin cyclohydrolase (Mch) is an enzyme involved in methanogenesis from CO₂ and H₂ which represents the energy metabolism of Methanopyrus kandleri, a methanogenic Archaeon growing at a temperature optimum of 98°C. The gene mch from M. kandleri was cloned, sequenced, and expressed in Escherichia coli. The overproduced enzyme could be purified in yields above 90% in one step by chromatography on phenyl Sepharose in 80% ammonium sulfate. From 3.5 g cells (250 mg protein), approximately 18 mg cyclohydrolase was obtained. The purified enzyme showed essentially the same catalytic properties as the enzyme purified from M. kandleri cells. The primary structure and properties of the cyclohydrolase are compared with those of the enzyme from Methanococcus jannaschii (growth temperature optimum 85°C), from Methanobacterium thermoautotrophicum (65°C), and from Methanosarcina barkeri (37°C). Of the four enzymes, that from M. kandleri has the lowest isoelectric point (3.8) and the lowest hydrophobicity of amino acid composition. Besides, it has the highest relative content of glutamate, leucine, and valine and the lowest relative content of isoleucine, serine, and lysine. Some of these properties are unusual for enzymes from hyperthermophilic organisms. They may reflect the observation that the cyclohydrolase from M. kandleri is not only adapted to hyperthermophilic conditions but also to the high intracellular concentrations of lyotrophic salts prevailing in this organism. Received: July 14, 1997 / Accepted: August 28, 1997     

Structural and functional analysis of SleL,a peptidoglycan lysin involved in germination of Bacillus spores

A major event in the germination of Bacillus spores concerns hydrolysis of the cortical peptidoglycan that surrounds the spore protoplast, the integrity of which is essential for maintenance of dormancy. Cortex degradation is initiated in all species of Bacillus spores by the combined activity of two semi‐redundant cortex‐lytic enzymes, SleB and CwlJ. A third enzyme, SleL, which has N‐acetylglucosaminidase activity, cleaves peptidoglycan fragments generated by SleB and CwlJ. Here we present crystal structures of B. cereus and B. megaterium SleL at 1.6 angstroms and 1.7 angstroms, respectively. The structures were determined with a view to identifying the structural basis of differences in catalytic efficiency between the respective enzymes. The catalytic (α/β)₈‐barrel cores of both enzymes are highly conserved from a structural perspective, including the spatial distribution of the catalytic residues. Both enzymes are equipped with two N‐terminal peptidoglycan‐binding LysM domains, which are also structurally highly conserved. However, the topological arrangement of the respective enzymes second LysM domain is markedly different, and this may account for differences in catalytic rates by impacting upon the position of the active sites with respect to their substrates. A chimeric enzyme comprising the B. megaterium SleL catalytic domain plus B. cereus SleL LysM domains displayed enzymatic activity comparable to the native B. cereus protein, exemplifying the importance of the LysM domains to SleL function. Similarly, the reciprocal construct, comprising the B. cereus SleL catalytic domain with B. megaterium SleL LysM domains, showed reduced activity compared with native B. cereus SleL. Proteins 2015; 83:1787–1799. © 2015 Wiley Periodicals, Inc.     

Primary structure and properties of the formyltransferase from the mesophilic Methanosarcina barkeri: comparison with the enzymes from thermophilic and hyperthermophilic methanogens

The ftr gene encoding formylmethanofuran: tetrahydromethanopterin formyltransferase (Ftr) from Methanosarcina barkeri was cloned, sequenced, and functionally expressed in Escherichia coli. The overproduced enzyme was purified eightfold to apparent homogeneity, and its catalytic properties were determined. The primary structure and the hydropathic character of the formyltransferase from Methanosarcina barkeri were compared with those of the enzymes from Methanobacterium thermoautotrophicum, Methanothermus fervidus, and Methanopyrus kandleri. The amino acid sequence of the enzyme from Methanosarcina barkeri was 64%, 61%, and 59% identical to that of the enzyme from Methanobacterium thermoautotrophicum, Methanothermus fervidus, and Methanopyrus kandleri, respectively. A negative correlation between the hydrophobicity of the enzymes and both the growth temperature optimum and the intracellular salt concentration of the four organisms was observed. The hydrophobicity of amino acid composition was +21.6 for the enzyme from Methanosarcina barkeri (growth temperature optimum 37° C, intracellular salt concentration ≈ 0.3 M), +9.9 for the enzyme from Methanobacterium thermoautotrophicum (65°C, ≈ 0.7 M), –20.8 for the enzyme from Methanothermus fervidus (83° C, ≈ 1.0 M) and –31.4 for the enzyme from Methanopyrus kandleri (98° C, > 1.1 M). Generally, a positive correlation between hydrophobicity and thermophilicity of enzymes and a negative correlation between hydrophobicity and halophilicity of enzymes are observed. The findings therefore indicate that the hydropathic character of the formyltransferases compared is mainly determined by the intracellular salt concentration rather than by temperature. Sequence similarities between the formyltransferases from methanogens and an open reading frame from Methylobacterium extorquens AM1 are discussed. Received: 7 September 1995 / Accepted: 7 November 1995     

Crystallographic and mutational studies on the tRNA thiouridine synthetase TtuA

In thermophilic bacteria, specific 2‐thiolation occurs on the conserved ribothymidine at position 54 (T54) in tRNAs, which is necessary for survival at high temperatures. T54 2‐thiolation is achieved by the tRNA thiouridine synthetase TtuA and sulfur‐carrier proteins. TtuA has five conserved CXXC/H motifs and the signature PP motif, and belongs to the TtcA family of tRNA 2‐thiolation enzymes, for which there is currently no structural information. In this study, we determined the crystal structure of a TtuA homolog from the hyperthermophilic archeon Pyrococcus horikoshii at 2.1 Å resolution. The P. horikoshii TtuA forms a homodimer, and each subunit contains a catalytic domain and unique N‐ and C‐terminal zinc fingers. The catalytic domain has much higher structural similarity to that of another tRNA modification enzyme, TilS (tRNA^Ile₂ lysidine synthetase), than to the other type of tRNA 2‐thiolation enzyme, MnmA. Three conserved cysteine residues are clustered in the putative catalytic site, which is not present in TilS. An in vivo mutational analysis in the bacterium Thermus thermophilus demonstrated that the three conserved cysteine residues and the putative ATP‐binding residues in the catalytic domain are important for the TtuA activity. A positively charged surface that includes the catalytic site and the two zinc fingers is likely to provide the tRNA‐binding site. Proteins 2013; 81:1232–1244. © 2013 Wiley Periodicals, Inc.     

Mutagenesis of tyrosine residues within helix VII in subunit I of the cytochrome <Emphasis Type="Italic">cbb</Emphasis><Subscript>3</Subscript> oxidase from <Emphasis Type="Italic">Rhodobacter capsulatus</Emphasis>

The cbb ₃-type oxidases are members of the heme-copper oxidase superfamily, distant by sequence comparisons, but sharing common functional characteristics. The cbb ₃ oxidases are missing an active-site tyrosine residue that is absolutely conserved in all A and B-type heme-copper oxidases. This tyrosine is known to play a critical role in the catalytic mechanisms of A and B-type oxidases. The absence of this tyrosine in the cbb ₃ oxidases raises the possibility that the cbb ₃ oxidases utilize a different catalytic mechanism from that of the other members of the superfamily, or have this conserved residue in different helices. Recently sequence comparisons indicate that, a tyrosine residues that might be analogous to the active-site tyrosine in other oxidases are present in the cbb ₃ oxidases but these tyrosines originates from a different transmembrane helix within the protein. In this research, three conserved tyrosine residues, Y294, Y308 and Y318, in helix VII were substituted for phenylalanine. Y318F mutant in the Rhodobacter capsulatus oxidase resulted in a fully assembled enzyme with nativelike structure and activity, but Y294F mutant is not assembled and have a catalytic activity. On the other hand, Y308F mutant is fully assembled enzyme with nativelike structure, but lacking catalytic activity. This result indicates that Y308 should be crucial in catalytic activity of the cbb ₃ oxidase of R. capsulatus. These findings support the assumption that all of the heme-copper oxidases utilize the same catalytic mechanism and provide a residue originates from different places within the primary sequence for different members of the same superfamily.     

Dynamics of the active site loops in catalyzing aminoacylation reaction in seryl and histidyl tRNA synthetases

Aminoacylation reaction is the first step of protein biosynthesis. The catalytic reorganization at the active site of aminoacyl tRNA synthetases (aaRSs) is driven by the loop motions. There remain lacunae of understanding concerning the catalytic loop dynamics in aaRSs. We analyzed the functional loop dynamics in seryl tRNA synthetase from Methanopyrus kandleri (^mkSerRS) and histidyl tRNA synthetases from Thermus thermophilus (^ttHisRS), respectively, using molecular dynamics. Results confirm that the motif 2 loop and other active site loops are flexible spots within the catalytic domain. Catalytic residues of the loops form a network of interaction with the substrates to form a reactive state. The loops undergo transitions between closed state and open state and the relaxation of the constituent residues occurs in femtosecond to nanosecond time scale. Order parameters are higher for constituent catalytic residues which form a specific network of interaction with the substrates to form a reactive state compared to the Gly residues within the loop. The development of interaction is supported from mutation studies where the catalytic domain with mutated loop exhibits unfavorable binding energy with the substrates. During the open-close motion of the loops, the catalytic residues make relaxation by ultrafast librational motion as well as fast diffusive motion and subsequently relax rather slowly via slower diffusive motion. The Gly residues act as a hinge to facilitate the loop closing and opening by their faster relaxation behavior. The role of bound water is analyzed by comparing implicit solvent-based and explicit solvent-based simulations. Loops fail to form catalytically competent geometry in absence of water. The present result, for the first time reveals the nature of the active site loop dynamics in aaRS and their influence on catalysis.     

玉米ZmPHO2基因家族克隆及其低磷胁迫下的表达

PHO2(编码一个泛素结合酶E2)作为磷高亲和转运体PHT1的负调控子,在维持植物体内磷的动态平衡中发挥重要作用。该研究以拟南芥和水稻中的PHO2为基础,从玉米自交系B73基因组中鉴定出9个ZmPHO2基因家族成员,在系统进化关系上将其分为3类。在玉米自交系178中克隆了上述9个基因的CDS全长序列,保守结构域分析发现,ZmPHO2蛋白质序列中均有1个由约130个氨基酸组成的泛素结合酶E2催化结构域(UBCc),其中包含1个重要的保守氨基酸(半胱氨酸)。实时荧光定量结果表明,低磷胁迫处理后,所有ZmPHO2基因均有表达,并呈现不同的表达模式,主要表现为叶与根之间的组织差异和玉米自交系178与9782之间的基因型差异,而在同一组织多数基因间的表达差异不明显。其中,ZmPHO2;H2在自交系9782的根中持续下调表达,但在叶中持续上调表达,表明ZmPHO2;H2可能参与调控磷素在叶与根之间的运输,以维持地上部分和地下部分磷的平衡。     

Bifunctional structure of two adenylyl cyclases from the myxobacterium Stigmatella aurantiaca

Two adenylyl cyclase genes (cyaA and cyaB) from the myxobacterium Stigmatella aurantiaca were cloned by complementation of Escherichia coli mutants defective in the cya gene. cyaA codes for a protein of 424 amino acid residues (AC1), while cyaB encodes a protein of 352 residues (AC2). Both cyclases are sensitive to adenosine: cAMP production was strongly inhibited in E coli cells and cell extracts expressing these genes. AC1 comprises a hydrophobic domain of six transmembrane helices coupled to a cytoplasmic catalytic domain endowed with adenylyl cyclase activity. A 17 amino acid residue sequence, which is a signature of G-protein coupled receptors, as well as of slime mold Dictyostelium discoideum cyclic AMP receptors, was found in the membrane domain. AC2 displays features also indicating that it is a bifunctional enzyme. The domain located upstream from the catalytic adenylyl cyclase domain shows strong similarity to receiver modules of response regulators of two-component bacterial signaling systems. In vitro mutagenesis of conserved aspartate residues in this domain was shown to interfere with cAMP synthesis.     

The cyclooxygenases

Cyclooxygenases (COXs) catalyze the rate-limiting step in the production of prostaglandins, bioactive compounds involved in processes such as fever and sensitivity to pain, and are the target of aspirin-like drugs. COX genes have been cloned from coral, tunicates and vertebrates, and in all the phyla where they are found, there are two genes encoding two COX isoenzymes; it is unclear whether these genes arose from an early single duplication event or from multiple independent duplications in evolution. The intron-exon arrangement of COX genes is completely conserved in vertebrates and mostly conserved in all species. Exon boundaries largely define the four functional domains of the encoded protein: the amino-terminal hydrophobic signal peptide, the dimerization domain, the membrane-binding domain, and the catalytic domain. The catalytic domain of each enzyme contains distinct peroxidase and cyclooxygenase active sites; COXs are classified as members of the myeloperoxidase family. All COXs are homodimers and monotopic membrane proteins (inserted into only one leaflet of the membrane), and they appear to be targeted to the lumenal membrane of the endoplasmic reticulum, where they are N-glycosylated. In mammals, the two COX genes encode a constitutive isoenzyme (COX-1) and an inducible isoenzyme (COX-2); both are of significant pharmacological importance.     

Isolation of a gene from Leuconostoc citreum B/110-1-2 encoding a novel dextransucrase enzyme

The amplicon encoding dextransucrase DSR-F from Leuconostoc citreum B/110-1-2, a novel sucrose glucosyltransferase (GTF)-specific for α-1,6 and α-1,3 glucosidic bond synthesis, with α-1,4 branching was cloned, sequenced, and expressed into Escherichia coli JM109. Recombinant enzyme catalyzed oligosaccharides synthesis from sucrose as donor and maltose acceptor. The dsrF gene encodes for a protein (DSR-F) of 1,528 amino acids, with a theoretical molecular mass of 170447.72 Da (~170 kDa). From amino acid sequence comparison, it appears that DSR-F possesses the same domains as those described for GTFs. However, the variable region is longer than in other GTFs (by 100 amino acids) and two APY repeats (a 79 residue long motif with a high number of conserved glycine and aromatic residues, characterized by the presence of the three consecutive residues Ala, Pro, and Tyr) were identified in the glucan binding domain. The DSR-F catalytic domain possesses the catalytic triad involved in the glucosyl enzyme formation. The amino acid sequence of this domain shares a 56% identity with catalytic domain of the alternansucrase ASR from L. citreum NRRL B-1355 and with the catalytic domain of a putative alternansucrase sequence found in the genome of L. citreum KM20. A truncated active variant DSR-F-∆SP-∆GBD of 1,251 amino acids, with a molecular mass of 145 544 Da (~145 kDa), was obtained.     

X-ray structure and mechanism of ZgHAD,a l-2-haloacid dehalogenase from the marine Flavobacterium Zobellia galactanivorans

Haloacid dehalogenases are potentially involved in bioremediation of contaminated environments and few have been biochemically characterized from marine organisms. The l -2-haloacid dehalogenase (l -2-HAD) from the marine Bacteroidetes Zobellia galactanivorans Dsij^T (ZgHAD) has been shown to catalyze the dehalogenation of C2 and C3 short-chain l -2-haloalkanoic acids. To better understand its catalytic properties, its enzymatic stability, active site, and 3D structure were analyzed. ZgHAD demonstrates high stability to solvents and a conserved catalytic activity when heated up to 60°C, its melting temperature being at 65°C. The X-ray structure of the recombinant enzyme was solved by molecular replacement. The enzyme folds as a homodimer and its active site is very similar to DehRhb, the other known l -2-HAD from a marine Rhodobacteraceae. Marked differences are present in the putative substrate entrance sites of the two enzymes. The H179 amino acid potentially involved in the activation of a catalytic water molecule was confirmed as catalytic amino acid through the production of two inactive site-directed mutants. The crystal packing of 13 dimers in the asymmetric unit of an active-site mutant, ZgHAD-H179N, reveals domain movements of the monomeric subunits relative to each other. The involvement of a catalytic His/Glu dyad and substrate binding amino acids was further confirmed by computational docking. All together our results give new insights into the catalytic mechanism of the group of marine l -2-HAD.     

Structural modeling and electrostatic properties of aspartate transcarbamylase from Saccharomyces cerevisiae

In Saccharomyces cerevisiae the first two reactions of the pyrimidine pathway are catalyzed by a multifunctional protein which possesses carbamylphosphate synthetase and aspartate transcarbamylase activities. Genetic and proteolysis studies suggested that the ATCase activity is carried out by an independently folded domain. In order to provide structural information for ongoing mutagenesis studies, a model of the three-dimensional structure of this domain was generated on the basis of the known X-ray structure of the related catalytic subunit from E. coli ATCase. First, a model of the catalytic monomer was built and refined by energy minimization. In this structure, the conserved residues between the two proteins were found to constitute the hydrophobic core whereas almost all the mutated residues are located at the surface. Then, a trimeric structure was generated in order to build the active site as it lies at the interface between adjacent chains in the E. coli catalytic trimer. After docking a bisubstrate analog into the active site, the whole structure was energy minimized to regularize the interactions at the contact areas between subunits. The resulting model is very similar to that obtained for the E. coli catalytic trimer by X-ray crystallography, with a remarkable conservation of the structure of the active site and its vicinity. Most of the interdomain and intersubunit interactions that are essential for the stability of the E. coli catalytic trimer are maintained in the yeast enzyme even though there is only 42% identity between the two sequences. Free energy calculations indicate that the trimeric assembly is more stable than the monomeric form. Moreover an insertion of four amino acids is localized in a loop which, in E. coli ATCase, is at the surface of the protein. This insertion exposes hydrophobic residues to the solvent. Interestingly, such an insertion is present in all the eukaryotic ATCase genes sequenced so far, suggesting that this region is interacting with another domain of the multifunctional protein. © 1994 Wiley-Liss, Inc.     

Expression, Purification, Characterization, and X-Ray Analysis of Selenomethionine 215 Variant of Leukocyte Collagenase

Matrix metalloproteinases belong to the superfamily of metzincins containing, besides a similar topology and a strictly conserved zinc environment, a 1,4-tight turn with a strictly conserved methionine residue at position three (the so called Met-turn [Bode et al. (1993) FEBS 331, 134–140; Stöcker et al. (1995) Protein Sci. 4, 823–840]. The distal S–CH₃ moiety of this methionine residue forms the hydrophobic basement of the three His residues liganding the catalytic zinc ion. To assess the importance of this methionine, we have expressed the catalytic domain of neutrophil collagenase (rHNC, residues Met80–Gly242) in the methionine auxotrophic Escherichia coli strain B834[DE3](hsd metB), with the two methionine residues replaced by Selenomethionine. Complete replacement was confirmed by amino acid analysis and electrospray mass spectrometry. The folded and purified enzyme retained its catalytic activity, but showed modifications which are reflected in changed kinetic parameters. The Met215SeMet substitution caused a decrease in conformational stability upon urea denaturation. The X-ray crystal structure of this Selenomethionine rHNC was virtually identical to that of the wild-type catalytic domain except for a very faint local disturbance around the sulfur-seleno substitution site.     

Tertiary structure of bacterial selenocysteine tRNA

Selenocysteine (Sec) is translationally incorporated into proteins in response to the UGA codon. The tRNA specific to Sec (tRNA^Sec) is first ligated with serine by seryl-tRNA synthetase (SerRS). In the present study, we determined the 3.1 Å crystal structure of the tRNA^Sec from the bacterium Aquifex aeolicus, in complex with the heterologous SerRS from the archaeon Methanopyrus kandleri. The bacterial tRNA^Sec assumes the L-shaped structure, from which the long extra arm protrudes. Although the D-arm conformation and the extra-arm orientation are similar to those of eukaryal/archaeal tRNA^Secs, A. aeolicus tRNA^Sec has unique base triples, G14:C21:U8 and C15:G20a:G48, which occupy the positions corresponding to the U8:A14 and R15:Y48 tertiary base pairs of canonical tRNAs. Methanopyrus kandleri SerRS exhibited serine ligation activity toward A. aeolicus tRNA^Sec in vitro. The SerRS N-terminal domain interacts with the extra-arm stem and the outer corner of tRNA^Sec. Similar interactions exist in the reported tRNA^Ser and SerRS complex structure from the bacterium Thermus thermophilus. Although the catalytic C-terminal domain of M. kandleri SerRS lacks interactions with A. aeolicus tRNA^Sec in the present complex structure, the conformational flexibility of SerRS is likely to allow the CCA terminal region of tRNA^Sec to enter the SerRS catalytic site.     

Functional and structural studies of pullulanase from Anoxybacillus sp. LM18‐11

Pullulanase is a debranching enzyme that specifically hydrolyzes the α‐1,6 glycosidic linkage of α‐glucans, and has wide industrial applications. Here, we report structural and functional studies of a new thermostable pullulanase from Anoxybacillus sp. LM18‐11 (PulA). Based on the hydrolysis products, PulA was classified as a type I pullulanase. It showed maximum activity at 60°C and pH 6.0. Kinetic study showed that the specific activity and K_m for pullulan of PulA are 750 U mg^?1 and 16.4 μmol L^?1, respectively. PulA has a half‐life of 48 h at 60°C. The remarkable thermostability makes PulA valuable for industrial usage. To further investigate the mechanism of the enzyme, we solved the crystal structures of PulA and its complexes with maltotriose and maltotetraose at 1.75–2.22 Å resolution. The PulA structure comprises four domains (N1, N2, A, and C). A is the catalytic domain, in which three conserved catalytic residues were identified (D413, E442, and D526). Two molecules of oligosaccharides were seen in the catalytic A domain in a parallel binding mode. Interestingly, another two oligosaccharides molecules were found between the N1 domain and the loop between the third β‐strand and the third α‐helix in the A domain. Based on sequence alignment and the ligand binding pattern, the N1 domain is identified as a new type of carbohydrate‐binding motif and classified to the CBM68 family. The structure solved here is the first structure of pullulanase which has carbohydrate bound to the N1 domain. Proteins 2014; 82:1685–1693. © 2013 Wiley Periodicals, Inc.     

The <Emphasis Type="Italic">Metarhizium anisopliae trp1</Emphasis> Gene: Cloning and Regulatory Analysis

The trp1 gene from the entomopathogenic fungus Metarhizium anisopliae, cloned by heterologous hybridization with the plasmid carrying the trpC gene from Aspergillus nidulans, was sequence characterized. The predicted translation product has the conserved catalytic domains of glutamine amidotransferase (G domain), indoleglycerolphosphate synthase (C domain), and phosphoribosyl anthranilate isomerase (F domain) organized as NH₂–G–C–F–COOH. The ORF is interrupted by a single intron of 60 nt that is position conserved in relation to trp genes from Ascomycetes and length conserved in relation to Basidiomycetes species. RT-PCR analysis suggests constitutive expression of trp1 gene in M. anisopliae.     

Differences and similarities in enzymes from the neopullulanase subfamily isolated from thermophilic species

Six glycoside hydrolase (GH) family 13 members, classified under the polyspecific neopullulanase subfamily GH13_20 (also termed cyclomaltodextrinase) were analysed. They originate from thermophilic bacterial strains (Anoxybacillus flavithermus, Laceyella sacchari, and Geobacillus thermoleovorans) or from environmental DNA, collected after in situ enrichments in Icelandic hot springs. The genes were isolated following the CODEHOP consensus primer strategy, utilizing the first two of the four conserved sequence regions in GH13. The typical domain structure of GH13_20, including an N-terminal domain (classified as CBM34), the catalytic module composed of the A-and B-domains, and a C-terminal domain, was found in five of the encoded enzymes (abbreviated Amy1, 89, 92, 98 and 132). These five enzymes degraded cyclomaltodextrins (CDs) and starch, while only three, Amy92 (L. sacchari), Amy98 (A. flavithermus) and Amy132 (environmental DNA), also harboured neopullulanase activity. The L. sacchari enzyme was monomeric, but with CD as the preferred substrate, which is an unusual combination. The sixth enzyme (Amy29 from environmental DNA), was composed of the ABC-domains only. Preferred substrate for Amy29 was pullulan, which was degraded to panose, and the enzyme had no detectable activity on CDs. In addition to its different activity profile and domain composition, Amy29 also displayed a different conservation (LPKF) in the fifth conserved region (MPKL) proposed to identify the subfamily. All enzymes had apparent temperature optima in the range 50–65°C, while thermostability varied, and was highest for Amy29 with a half-life of 480 min at 80°C. Calcium dependent activity or stability was monitored in four enzymes, but could not be detected for Amy29 or 98. Tightly bound calcium can, however, not be ruled out, and putative calcium ligands were conserved in Amy98.     

Two Clip Domain Family of Serine Proteases and a Serine Protease Homolog from the Fall Webworm, Hyphantria cunea; cDNA Cloning and Characterization

Two types of serine proteases and a serine protease homologue cDNAs were isolated from Hyphantria cunea larvae induced immune response due to an injection of a microorganism through RT‐PCR and cDNA library screening, and their characteristics were examined. The isolated cDNAs are composed 2.1 kb, 2.2 kb, and 2.5 kb nucleotide each, which encoded 388, 390, 580 amino acid residues, and were designated as HcPE‐1, HcPE‐2 and HcPE‐3, respectively. They were revealed as serine proteases or a serine protease homologue with the clip domain through a database search. The deduced amino acid sequence comparison showed high homology of 72‐78% among them. Six Cys residues of the N‐terminal clip domain forming the disulfide bond, Cys residues of the catalytic domain, and Cys residues forming inter‐bridge between clip domain and catalytic domain were also well preserved. Three amino acid residues, His, Asp, and Ser, within the active site were perfectly conserved in HcPE‐2 and HcPE‐3, however, His was replaced with Gln¹⁷⁸ in HcPE‐1. The Arg residues (HcPE‐1, Arg¹³²; HcPE‐2, Arg¹³⁴; HcPE‐3, Arg³²⁵) known as the activation sites by proteolytic cleavage were preserved well in all three types of protein. In case of HcPE‐3, three continuous clip‐like domains existed in the N terminal. As the result of phylogenetic analysis, three clip domain family of protein from H. cunea make groups with arthropod proclotting enzyme precursor. Northern blot analysis showed all three genes were induced through an injection of Escherichia coli, but expression patterns were varied.     

Structure of N-acetyl-beta-D-glucosaminidase (GcnA) from the endocarditis pathogen Streptococcus gordonii and its complex with the mechanism-based inhibitor NAG-thiazoline

The crystal structure of GcnA, an N-acetyl-β-d-glucosaminidase from Streptococcus gordonii, was solved by multiple wavelength anomalous dispersion phasing using crystals of selenomethionine-substituted protein. GcnA is a homodimer with subunits each comprised of three domains. The structure of the C-terminal α-helical domain has not been observed previously and forms a large dimerisation interface. The fold of the N-terminal domain is observed in all structurally related glycosidases although its function is unknown. The central domain has a canonical (β/α)₈ TIM-barrel fold which harbours the active site. The primary sequence and structure of this central domain identifies the enzyme as a family 20 glycosidase. Key residues implicated in catalysis have different conformations in two different crystal forms, which probably represent active and inactive conformations of the enzyme. The catalytic mechanism for this class of glycoside hydrolase, where the substrate rather than the enzyme provides the cleavage-inducing nucleophile, has been confirmed by the structure of GcnA complexed with a putative reaction intermediate analogue, N-acetyl-β-d-glucosamine-thiazoline. The catalytic mechanism is discussed in light of these and other family 20 structures.