Effect of Dimer Dissociation on Activity and Thermostability of the α-Glucuronidase from Geobacillus stearothermophilus: Dissecting the Different Oligomeric Forms of Family 67 Glycoside Hydrolases

ABSTRACT The oligomeric organization of enzymes plays an important role in many biological processes, such as allosteric regulation, conformational stability and thermal stability. α-Glucuronidases are family 67 glycosidases that cleave the α-1,2-glycosidic bond between 4-O-methyl-d-glucuronic acid and xylose units as part of an array of hemicellulose-hydrolyzing enzymes. Currently, two crystal structures of α-glucuronidases are available, those from Geobacillus stearothermophilus (AguA) and from Cellvibrio japonicus (GlcA67A). Both enzymes are homodimeric, but surprisingly their dimeric organization is different, raising questions regarding the significance of dimerization for the enzymes' activity and stability. Structural comparison of the two enzymes suggests several elements that are responsible for the different dimerization organization. Phylogenetic analysis shows that the α-glucuronidases AguA and GlcA67A can be classified into two distinct subfamilies of bacterial α-glucuronidases, where the dimer-forming residues of each enzyme are conserved only within its own subfamily. It seems that the different dimeric forms of AguA and GlcA67A represent the two alternative dimeric organizations of these subfamilies. To study the biological significance of the dimerization in α-glucuronidases, we have constructed a monomeric form of AguA by mutating three of its interface residues (W328E, R329T, and R665N). The activity of the monomer was significantly lower than the activity of the wild-type dimeric AguA, and the optimal temperature for activity of the monomer was around 35°C, compared to 65°C of the wild-type enzyme. Nevertheless, the melting temperature of the monomeric protein, 72.9°C, was almost identical to that of the wild-type, 73.4°C. It appears that the dimerization of AguA is essential for efficient catalysis and that the dissociation into monomers results in subtle conformational changes in the structure which indirectly influence the active site region and reduce the activity. Structural and mechanistic explanations for these effects are discussed.

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Cell cycle arrest and astrocytic differentiation resulting from PTEN expression in glioma cells

Journal of Neurosurgery ◽

10.3171/jns.1999.91.5.0822 ◽

1999 ◽

Vol 91 (5) ◽

pp. 822-830 ◽

Cited By ~ 33

Author(s):

Jun-ichi Adachi ◽

Katsumi Ohbayashi ◽

Tomonari Suzuki ◽

Tomio Sasaki

Keyword(s):

Expression System ◽

Biological Significance ◽

Glioma Cells ◽

Genetic Alterations ◽

Pten Expression ◽

Human Glioma ◽

Wild Type ◽

Content Type ◽

Astrocytic Differentiation ◽

Fine Print

Object. Genetic alterations of the PTEN gene (also known as MMAC1 or TEP1) have frequently been identified in high-grade gliomas, indicating that inactivation of PTEN plays a crucial role in human glioma progression. The aim of this study was to assess the biological significance of PTEN inactivation in the development of glioma.Methods. The authors introduced wild-type PTEN complementary DNA into four human glioma cell lines (T98G, U-251MG, U-87MG, and A172) containing endogenous aberrant PTEN alleles. The number of colonies transfected with the wild-type PTEN was reduced to 15 to 32% of those found after transfection of a control vector, suggesting growth suppression by the exogenous PTEN. To analyze phenotypic alterations produced by PTEN expression, T98G-derived clones with inducible PTEN expression were further established using a tetracycline-regulated inducible gene expression system. Induction of PTEN expression suppressed the in vitro growth of T98G cells with accumulation of G1 phase cells. Furthermore, when cells were cultured in the presence of the extracellular matrix (ECM), PTEN expression caused distinct morphological changes, with multiple and elongated cytoplasmic processes similar to those of normal astrocytes. The level of glial fibrillary acidic protein, an intermediate protein specifically expressed in differentiated astrocytes, was upregulated concomitantly.Conclusions. These findings strongly indicate that exogenous PTEN expression inhibits the proliferation of glioma cells by inducing G1 arrest and elicits astrocytic differentiation in the presence of the ECM. Inactivation of PTEN would play an important role in the enhancement of unregulated growth of undifferentiated glioma cells.

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New Insights into the Spring-Loaded Conformational Change of Influenza Virus Hemagglutinin

Journal of Virology ◽

10.1128/jvi.76.9.4456-4466.2002 ◽

2002 ◽

Vol 76 (9) ◽

pp. 4456-4466 ◽

Cited By ~ 42

Author(s):

Jennifer A. Gruenke ◽

R. Todd Armstrong ◽

William W. Newcomb ◽

Jay C. Brown ◽

Judith M. White

Keyword(s):

Influenza Virus ◽

Conformational Change ◽

Conformational Changes ◽

Limited Proteolysis ◽

Coiled Coil ◽

Fusion Peptide ◽

Wild Type ◽

Influenza Virus Hemagglutinin ◽

Target Membrane ◽

Mutant Forms

ABSTRACT Influenza virus hemagglutinin undergoes a conformational change in which a loop-to-helix “spring-loaded” conformational change forms a coiled coil that positions the fusion peptide for interaction with the target bilayer. Previous work has shown that two proline mutations designed to disrupt this change disrupt fusion but did not determine the basis for the fusion defect. In this work, we made six additional mutants with single proline substitutions in the region that undergoes the spring-loaded conformational change and two additional mutants with double proline substitutions in this region. All double mutants were fusion inactive. We analyzed one double mutant, F63P/F70P, as an example. We observed that F63P/F70P undergoes key low-pH-induced conformational changes and binds tightly to target membranes. However, limited proteolysis and electron microscopy observations showed that the mutant forms a coiled coil that is only ∼50% the length of the wild type, suggesting that it is splayed in its N-terminal half. This work further supports the hypothesis that the spring-loaded conformational change is necessary for fusion. Our data also indicate that the spring-loaded conformational change has another role beyond presenting the fusion peptide to the target membrane.

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Surface-Exposed Adeno-Associated Virus Vp1-NLS Capsid Fusion Protein Rescues Infectivity of Noninfectious Wild-Type Vp2/Vp3 and Vp3-Only Capsids but Not That of Fivefold Pore Mutant Virions

Journal of Virology ◽

10.1128/jvi.00580-07 ◽

2007 ◽

Vol 81 (15) ◽

pp. 7833-7843 ◽

Cited By ~ 43

Author(s):

Joshua C. Grieger ◽

Jarrod S. Johnson ◽

Brittney Gurda-Whitaker ◽

Mavis Agbandje-McKenna ◽

R. Jude Samulski

Keyword(s):

Conformational Changes ◽

Structural Changes ◽

Wild Type ◽

Dot Blot ◽

Adeno Associated Virus ◽

N Terminus ◽

Localization Signal ◽

Significant Effort

ABSTRACT Over the past 2 decades, significant effort has been dedicated to the development of adeno-associated virus (AAV) as a vector for human gene therapy. However, understanding of the virus with respect to the functional domains of the capsid remains incomplete. In this study, the goal was to further examine the role of the unique Vp1 N terminus, the N terminus plus the recently identified nuclear localization signal (NLS) (J. C. Grieger, S. Snowdy, and R. J. Samulski, J. Virol 80:5199-5210, 2006), and the virion pore at the fivefold axis in infection. We generated two Vp1 fusion proteins (Vp1 and Vp1NLS) linked to the 8-kDa chemokine domain of rat fractalkine (FKN) for the purpose of surface exposure upon assembly of the virion, as previously described (K. H. Warrington, Jr., O. S. Gorbatyuk, J. K. Harrison, S. R. Opie, S. Zolotukhin, and N. Muzyczka, J. Virol 78:6595-6609, 2004). The unique Vp1 N termini were found to be exposed on the surfaces of these capsids and maintained their phospholipase A2 (PLA2) activity, as determined by native dot blot Western and PLA2 assays, respectively. Incorporation of the fusions into AAV type 2 capsids lacking a wild-type Vp1, i.e., Vp2/Vp3 and Vp3 capsid only, increased infectivity by 3- to 5-fold (Vp1FKN) and 10- to 100-fold (Vp1NLSFKN), respectively. However, the surface-exposed fusions did not restore infectivity to AAV virions containing mutations at a conserved leucine (Leu336Ala, Leu336Cys, or Leu336Trp) located at the base of the fivefold pore. EM analyses suggest that Leu336 may play a role in global structural changes to the virion directly impacting downstream conformational changes essential for infectivity and not only have local effects within the pore, as previously suggested.

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Molecular basis for defective secretion of the Z variant of human alpha-1-proteinase inhibitor: secretion of variants having altered potential for salt bridge formation between amino acids 290 and 342

Molecular and Cellular Biology ◽

10.1128/mcb.9.4.1406-1414.1989 ◽

1989 ◽

Vol 9 (4) ◽

pp. 1406-1414

Author(s):

A A McCracken ◽

K B Kruse ◽

J L Brown

Keyword(s):

Amino Acid ◽

Proteinase Inhibitor ◽

Conformational Stability ◽

Point Mutations ◽

Salt Bridge ◽

Site Directed Mutagenesis ◽

Wild Type ◽

Functional Importance ◽

Bridge Formation ◽

Cos Cells

Human alpha-1-proteinase inhibitor (A1PI) deficiency, associated with the Z-variant A1PI (A1PI/Z) gene, results from defective secretion of the inhibitor from the liver. The A1PI/Z gene exhibits two point mutations which specify amino acid substitutions, Val-213 to Ala and Glu-342 to Lys. The functional importance of these substitutions in A1PI deficiency was investigated by studying the secretion of A1PI synthesized in COS cells transfected with A1PI genes altered by site-directed mutagenesis. This model system correctly duplicates the secretion defect seen in individuals homozygous for the A1PI/Z allele and shows that the substitution of Lys for Glu-342 alone causes defective secretion of A1PI. The substitution of Lys for Glu-342 eliminates the possibility for a salt bridge between residues 342 and 290, which may decrease the conformational stability of the molecule and thus account for the secretion defect. However, when we removed the potential to form a salt bridge from the wild-type inhibitor by changing Lys-290 to Glu (A1PI/SB-290Glu), secretion was not reduced to the 19% of normal level seen for A1PI/Z-342Lys; in fact, 75% of normal secretion was observed. When the potential for salt bridge formation was returned to A1PI/Z-342Lys by changing Lys-290 to Glu, only 46% of normal secretion was seen. These data indicate that the amino acid substitution at position 342, rather than the potential to form the 290-342 salt bridge, is the critical alteration leading to the defect in A1PI secretion.

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Genomic and Transcriptome Analyses of a Thermophilic Bacterium Geobacillus stearothermophilus B5 Isolated from Compost Reveal Its Enzymatic Basis for Lignocellulose Degradation

Microorganisms ◽

10.3390/microorganisms8091357 ◽

2020 ◽

Vol 8 (9) ◽

pp. 1357

Author(s):

Mengmeng Wang ◽

Jiaxi Miao ◽

Xuanqing Wang ◽

Tuo Li ◽

Han Zhu ◽

...

Keyword(s):

Thermophilic Bacterium ◽

Glycoside Hydrolases ◽

Lignocellulose Degradation ◽

Geobacillus Stearothermophilus ◽

Carbohydrate Active Enzymes ◽

Transcriptional Responses ◽

Protein Coding ◽

Clusters Of Orthologous Groups ◽

Different Temperatures ◽

Shock Proteins

A lignocellulose-degrading strain isolated from thermophilic compost was identified as Geobacillus stearothermophilus B5, and found able to secrete considerable amounts of enzymes at optimal temperature (60 °C) and pH (7.5). One circular contig of 3.37 Mbp was assembled from raw data, and 3371 protein-coding genes were predicted. Clusters of orthologous groups (COG) analysis revealed various genes with functions in polymeric substrate degradation, especially for Carbohydrate Active enZymes (CAZymes), such as glycoside hydrolases (GHs) and glycosyl transferases (GTs). Furthermore, the transcriptional responses of B5 at different temperatures—with rice straw provided as the sole carbon source—were analyzed. The results revealed that B5 could resist high temperature by upregulating heat shock proteins (HSPs), enhancing protein synthesis, and decreasing carbon catabolism. Briefly, B5 possesses the ability of lignocellulose degradation, and might be considered a potential inoculant for improving composting efficiency.

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Role of Flagellin-Homologous Proteins in Biofilm Formation by Pathogenic Vibrio Species

mBio ◽

10.1128/mbio.01793-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 2

Author(s):

You-Chul Jung ◽

Mi-Ae Lee ◽

Kyu-Ho Lee

Keyword(s):

Biofilm Formation ◽

Specific Binding ◽

Biological Significance ◽

Wild Type ◽

Forming Process ◽

Homologous Proteins ◽

Content Type ◽

Pathogenic Vibrio ◽

Biofilm Matrix

ABSTRACT The pathogenic bacterium Vibrio vulnificus exhibits the ability to form biofilm, for which initiation is dependent upon swimming motility by virtue of a polar flagellum. The filament of its flagellum is composed of multiple flagellin subunits, FlaA, -B, -C, and -D. In V. vulnificus genomes, however, open reading frames (ORFs) annotated by FlaE and -F are also present. Although neither FlaE nor FlaF is involved in filament formation and cellular motility, they are well expressed and secreted to the extracellular milieu through the secretion apparatus for flagellar assembly. In the extrapolymeric matrix of V. vulnificus biofilm, significant levels of FlaEF were detected. Mutants defective in both flaE and flaF formed significantly decreased biofilms compared to the wild-type biofilm. Thus, the potential role of FlaEF during the biofilm-forming process was investigated by exogenous addition of recombinant FlaEF (rFlaEF) to the biofilm assays. The added rFlaE and rFlaF were predominantly incorporated into the biofilm matrix formed by the wild type. However, biofilms formed by a mutant defective in exopolysaccharide (EPS) biosynthesis were not affected by added FlaEF. These results raised a possibility that FlaEF specifically interact with EPS within the biofilm matrix. In vitro pulldown assays using His-tagged rFlaEF or rFlaC revealed the specific binding of EPS to rFlaEF but not to rFlaC. Taken together, our results demonstrate that V. vulnificus FlaEF, flagellin-homologous proteins (FHPs), are crucial for biofilm formation by directly interacting with the essential determinant for biofilm maturation, EPS. Further analyses performed with other pathogenic Vibrio species demonstrated both the presence of FHPs and their important role in biofilm formation. IMPORTANCE Flagellar filaments of the pathogenic Vibrio species, including V. vulnificus, V. parahaemolyticus, and V. cholerae, are composed of multiple flagellin subunits. In their genomes, however, there are higher numbers of the ORFs encoding flagellin-like proteins than the numbers of flagellin subunits required for filament assembly. Since these flagellin-homologous proteins (FHPs) are well expressed and excreted to environments via a flagellin transport channel, their extracellular role in the pathogenic Vibrio has been enigmatic. Their biological significance, which is not related with flagellar functions, has been revealed to be in maturation of biofilm structures. Among various components of the extracellular polymeric matrix produced in the V. vulnificus biofilms, the exopolysaccharides (EPS) are dominant constituents and crucial in maturation of biofilms. The enhancing role of the V. vulnificus FHPs in biofilm formation requires the presence of EPS, as indicated by highly specific interactions among two FHPs and three EPS.

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P219L substitution in human D-amino acid oxidase impacts the ligand binding and catalytic efficiency

The Journal of Biochemistry ◽

10.1093/jb/mvaa083 ◽

2020 ◽

Vol 168 (5) ◽

pp. 557-567

Author(s):

Wanitcha Rachadech ◽

Yusuke Kato ◽

Rabab M Abou El-Magd ◽

Yuji Shishido ◽

Soo Hyeon Kim ◽

...

Keyword(s):

Amino Acid ◽

Conformational Changes ◽

Active Site ◽

Structural Changes ◽

Catalytic Efficiency ◽

Acid Oxidase ◽

Wild Type ◽

Amino Acid Oxidase ◽

Adenine Ring ◽

The Impact

Abstract Human D-amino acid oxidase (DAO) is a flavoenzyme that is implicated in neurodegenerative diseases. We investigated the impact of replacement of proline with leucine at Position 219 (P219L) in the active site lid of human DAO on the structural and enzymatic properties, because porcine DAO contains leucine at the corresponding position. The turnover numbers (kcat) of P219L were unchanged, but its Km values decreased compared with wild-type, leading to an increase in the catalytic efficiency (kcat/Km). Moreover, benzoate inhibits P219L with lower Ki value (0.7–0.9 µM) compared with wild-type (1.2–2.0 µM). Crystal structure of P219L in complex with flavin adenine dinucleotide (FAD) and benzoate at 2.25 Å resolution displayed conformational changes of the active site and lid. The distances between the H-bond-forming atoms of arginine 283 and benzoate and the relative position between the aromatic rings of tyrosine 224 and benzoate were changed in the P219L complex. Taken together, the P219L substitution leads to an increase in the catalytic efficiency and binding affinity for substrates/inhibitors due to these structural changes. Furthermore, an acetic acid was located near the adenine ring of FAD in the P219L complex. This study provides new insights into the structure–function relationship of human DAO.

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Effect of human acetylcholinesterase subunit assembly on its circulatory residence

Biochemical Journal ◽

10.1042/bj3540613 ◽

2001 ◽

Vol 354 (3) ◽

pp. 613-625 ◽

Cited By ~ 12

Author(s):

Theodor CHITLARU ◽

Chanoch KRONMAN ◽

Baruch VELAN ◽

Avigdor SHAFFERMAN

Keyword(s):

Laser Desorption Ionization ◽

Wild Type ◽

Ionization Time ◽

Hek 293 ◽

Enzyme Preparations ◽

Hek 293 Cells ◽

293 Cells ◽

Differential Behaviour ◽

Oligomeric Forms

Sialylated recombinant human acetylcholinesterase (rHuAChE), produced by stably transfected cells, is composed of a mixed population of monomers, dimers and tetramers and manifests a time-dependent circulatory enrichment of the higher-order oligomeric forms. To investigate this phenomenon further, homogeneous preparations of rHuAChE differing in their oligomerization statuses were generated: (1) monomers, represented by the oligomerization-impaired C580A-rHuAChE mutant, (2) wild-type (WT) dimers and (3) tetramers of WT-rHuAChE generated in vitro by complexation with a synthetic ColQ-derived proline-rich attachment domain (‘PRAD’) peptide. Three different series of each of these three oligoform preparations were produced: (1) partly sialylated, derived from HEK-293 cells; (2) fully sialylated, derived from engineered HEK-293 cells expressing high levels of sialyltransferase; and (3) desialylated, after treatment with sialidase to remove sialic acid termini quantitatively. The oligosaccharides associated with each of the various preparations were extensively analysed by matrix-assisted laser desorption ionization–time-of-flight MS. With the enzyme preparations comprising the fully sialylated series, a clear linear relationship between oligomerization and circulatory mean residence time (MRT) was observed. Thus monomers, dimers and tetramers exhibited MRTs of 110, 195 and 740min respectively. As the level of sialylation decreased, this differential behaviour became less pronounced; eventually, after desialylation all oligoforms had the same MRT (5min). These observations suggest that multiple removal systems contribute to the elimination of AChE from the circulation. Here we also demonstrate that by the combined modulation of sialylation and tetramerization it is possible to generate a rHuAChE displaying a circulatory residence exceeding that of all other known forms of native or recombinant human AChE.

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The Role of a P1-Type ATPase from Pseudomonas fluorescens SBW25 in Copper Homeostasis and Plant Colonization

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-20-5-0581 ◽

2007 ◽

Vol 20 (5) ◽

pp. 581-588 ◽

Cited By ~ 21

Author(s):

Xue-Xian Zhang ◽

Paul B. Rainey

Keyword(s):

Pseudomonas Fluorescens ◽

Mutant Strain ◽

Copper Ions ◽

Biological Significance ◽

Copper Homeostasis ◽

Plant Colonization ◽

Wild Type ◽

Pseudomonas Fluorescens Sbw25 ◽

Plant Surfaces

The genome of the plant-colonizing bacterium Pseudomonas fluorescens SBW25 possesses a putative copper-transporting P1-type ATPase (CueA) that is induced on the plant surfaces. Using a chromosomally-integrated cueA-'lacZ fusion, we show that transcription of cueA can be induced (in vitro) by ions of copper, silver, gold, and mercury. To investigate the biological significance of cueA, a nonpolar cueA deletion mutant (SBW25ΔcueA) was constructed. This mutant strain displayed a twofold reduction in its tolerance to copper compared with the wild-type strain; however, no change was observed in the sensitivity of the mutant strain to silver, gold, or mercury ions. To obtain insight into the ecological significance of cueA, the competitive ability of SBW25ΔcueA was determined relative to wild-type SBW25 in three environments (none contained added copper): minimal M9 medium, the root of sugar beet (Beta vulgaris), and the root of pea (Pisum sativum). Results showed that the fitness of SBW25ΔcueA was not different from the wild type in laboratory medium but was compromised in the two plant environments. Taken together, these data demonstrate a functional role for CueA in copper homeostasis and reveal an ecologically significant contribution to bacterial fitness in the plant rhizosphere. They also suggest that copper ions accumulate on plant surfaces.

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