scholarly journals beta-Glucosidase in cellulosome of the anaerobic fungus Piromyces sp. strain E2 is a family 3 glycoside hydrolase

2003 ◽  
Vol 370 (3) ◽  
pp. 963-970 ◽  
Author(s):  
Peter J.M. STEENBAKKERS ◽  
Harry R. HARHANGI ◽  
Mirjam W. BOSSCHER ◽  
Marlous M.C. van der HOOFT ◽  
Jan T. KELTJENS ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Glycoside Hydrolase ◽  
Catalytic Domain ◽  
Crystalline Cellulose ◽  
Anaerobic Fungi ◽  
Anaerobic Fungus ◽  
Glucosidase Activity ◽  
Biochemical Analyses ◽  
Beta Glucosidase

The cellulosomes of anaerobic fungi convert crystalline cellulose solely into glucose, in contrast with bacterial cellulosomes which produce cellobiose. Previously, a β-glucosidase was identified in the cellulosome of Piromyces sp. strain E2 by zymogram analysis, which represented approx. 25% of the extracellular β-glucosidase activity. To identify the component in the fungal cellulosome responsible for the β-glucosidase activity, immunoscreening with anti-cellulosome antibodies was used to isolate the corresponding gene. A 2737bp immunoclone was isolated from a cDNA library. The clone encoded an extracellular protein containing a eukaryotic family 3 glycoside hydrolase domain homologue and was therefore named cel3A. The C-terminal end of the encoded Cel3A protein consisted of an auxiliary domain and three fungal dockerins, typical for cellulosome components. The Cel3A catalytic domain was expressed in Escherichia coli BL21 and purified. Biochemical analyses of the recombinant protein showed that the Cel3A catalytic domain was specific for β-glucosidic bonds and functioned as an exoglucohydrolase on soluble substrates as well as cellulose. Comparison of the apparent Km and Ki values of heterologous Cel3A and the fungal cellulosome for p-nitrophenyl-β-d-glucopyranoside and d-glucono-1,5-Δ-lactone respectively indicated that cel3A encodes the β-glucosidase activity of the Piromyces sp. strain E2 cellulosome.

scholarly journals The modular architecture of Cellvibrio japonicus mannanases in glycoside hydrolase families 5 and 26 points to differences in their role in mannan degradation

2003 ◽  
Vol 371 (3) ◽  
pp. 1027-1043 ◽  
Author(s):  
Deborah HOGG ◽  
Gavin PELL ◽  
Paul DUPREE ◽  
Florence GOUBET ◽  
Susana M. MARTÍN-ORÚE ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Plant Cell Wall ◽  
Catalytic Domain ◽  
Glycoside Hydrolases ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Molecular Architecture ◽  
Catalytic Domains ◽  
Carbohydrate Binding Modules ◽  
Cellvibrio Japonicus

β-1,4-Mannanases (mannanases), which hydrolyse mannans and glucomannans, are located in glycoside hydrolase families (GHs) 5 and 26. To investigate whether there are fundamental differences in the molecular architecture and biochemical properties of GH5 and GH26 mannanases, four genes encoding these enzymes were isolated from Cellvibrio japonicus and the encoded glycoside hydrolases were characterized. The four genes, man5A, man5B, man5C and man26B, encode the mannanases Man5A, Man5B, Man5C and Man26B, respectively. Man26B consists of an N-terminal signal peptide linked via an extended serine-rich region to a GH26 catalytic domain. Man5A, Man5B and Man5C contain GH5 catalytic domains and non-catalytic carbohydrate-binding modules (CBMs) belonging to families 2a, 5 and 10; Man5C in addition contains a module defined as X4 of unknown function. The family 10 and 2a CBMs bound to crystalline cellulose and ivory nut crystalline mannan, displaying very similar properties to the corresponding family 10 and 2a CBMs from Cellvibrio cellulases and xylanases. CBM5 bound weakly to these crystalline polysaccharides. The catalytic domains of Man5A, Man5B and Man26B hydrolysed galactomannan and glucomannan, but displayed no activity against crystalline mannan or cellulosic substrates. Although Man5C was less active against glucomannan and galactomannan than the other mannanases, it did attack crystalline ivory nut mannan. All the enzymes exhibited classic endo-activity producing a mixture of oligosaccharides during the initial phase of the reaction, although their mode of action against manno-oligosaccharides and glucomannan indicated differences in the topology of the respective substrate-binding sites. This report points to a different role for GH5 and GH26 mannanases from C. japonicus. We propose that as the GH5 enzymes contain CBMs that bind crystalline polysaccharides, these enzymes are likely to target mannans that are integral to the plant cell wall, while GH26 mannanases, which lack CBMs and rapidly release mannose from polysaccharides and oligosaccharides, target the storage polysaccharide galactomannan and manno-oligosaccharides.

scholarly journals Characterization and structure of glyceraldehyde-3-phosphate dehydrogenase type 1 from Escherichia coli

2020 ◽  
Vol 76 (9) ◽  
pp. 406-413
Author(s):  
L. Zhang ◽  
M. R. Liu ◽  
Y. C. Yao ◽  
I. K. Bostrom ◽  
Y. D. Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Catalytic Domain ◽  
X Ray Diffraction ◽  
Cell Parameters ◽  
Diffusion Technique ◽  
Key Enzyme ◽  
Optimum Reaction ◽  
Biochemical Analyses

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in the glycolytic pathway that catalyzes the conversion of D-glyceraldehyde 3-phosphate to 1,3-diphosphoglycerate. Here, the full-length GAPDH type 1 from Escherichia coli (EcGAPDH1) was cloned and overexpressed, and the protein was purified. Biochemical analyses found that the optimum reaction temperature and pH of EcGAPDH1 were 55°C and 10.0, respectively. The protein has a certain amount of thermostability. Crystals of EcGAPDH1 were obtained using the sitting-drop vapor-diffusion technique and X-ray diffraction data were collected to 1.88 Å resolution. Characterization of the crystals showed that they belonged to space group P41212, with unit-cell parameters a = b = 89.651, c = 341.007 Å, α = β = γ = 90°. The structure of EcGAPDH1 contains four subunits, each of which includes an N-terminal NAD+-binding domain and a C-terminal catalytic domain. Analysis of the NAD+-bound form showed some differences between the structures of EcGAPDH1 and human GAPDH. As EcGAPDH1 shares 100% identity with GAPDH from Shigella sonnei, its structure may help in finding a drug for the treatment of shigellosis.

scholarly journals Intronless celB from the anaerobic fungus Neocallimastix patriciarum encodes a modular family A endoglucanase

1994 ◽  
Vol 297 (2) ◽  
pp. 359-364 ◽  
Author(s):  
L Zhou ◽  
G P Xue ◽  
C G Orpin ◽  
G W Black ◽  
H J Gilbert ◽  
...  
Keyword(s):  
Tandem Repeats ◽  
Catalytic Domain ◽  
Crystalline Cellulose ◽  
Anaerobic Fungus ◽  
Beta Glucan ◽  
Reading Frame ◽  
Neocallimastix Patriciarum ◽  
Rumen Fungus ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of

The cDNA designated celB from the anaerobic rumen fungus Neocallimastix patriciarum contained a single open reading frame of 1422 bp coding for a protein (CelB) of M(r) 53,070. CelB expressed by Escherichia coli harbouring the full-length gene hydrolysed carboxymethylcellulose in the manner of an endoglucanase, but was most active against barley beta-glucan. It also released reducing sugar from xylan and lichenan, but was inactive against crystalline cellulose, laminarin, mannan, galactan and arabinan. The rate of hydrolysis of cellulo-oligosaccharides by CelB increased with increasing chain length from cellotriose to cellopentaose. The predicted structure of CelB contained features indicative of modular structure. The first 360 residues of CelB constituted a fully functional catalytic domain that was homologous with bacterial endoglucanases belonging to cellulase family A, including five which originate from three different species of anaerobic rumen bacteria. Downstream from this domain, and linked to it by a serine/threonine-rich hinge, was a non-catalytic domain containing short tandem repeats, homologous to the C-terminal repeats contained in xylanase A from the same anaerobic fungus. Unlike previous fungal cellulases, genomic celB was devoid of introns. This lack of introns and the homology of its encoded product with rumen bacterial endoglucanases suggest that acquisition of celB by the fungus may at some stage have involved horizontal gene transfer from a prokaryote to N. particiarum.

Expression of hepatitis A virus cDNA in Escherichia coli: antigenic VP1 recombinant protein.

1987 ◽  
Vol 61 (11) ◽  
pp. 3645-3647 ◽  
Author(s):  
R Ostermayr ◽  
K von der Helm ◽  
V Gauss-Müller ◽  
E L Winnacker ◽  
F Deinhardt
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Virus Cdna

scholarly journals Functional Studies of [FeFe] Hydrogenase Maturation in an Escherichia coli Biosynthetic System

2006 ◽  
Vol 188 (6) ◽  
pp. 2163-2172 ◽  
Author(s):  
Paul W. King ◽  
Matthew C. Posewitz ◽  
Maria L. Ghirardi ◽  
Michael Seibert
Keyword(s):  
Escherichia Coli ◽  
Catalytic Domain ◽  
Expression System ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
E Coli ◽  
Functional Studies ◽  
Hydrogenase Maturation ◽  
Iron Sulfur ◽  
And Function

ABSTRACT Maturation of [FeFe] hydrogenases requires the biosynthesis and insertion of the catalytic iron-sulfur cluster, the H cluster. Two radical S-adenosylmethionine (SAM) proteins proposed to function in H cluster biosynthesis, HydEF and HydG, were recently identified in the hydEF-1 mutant of the green alga Chlamydomonas reinhardtii (M. C. Posewitz, P. W. King, S. L. Smolinski, L. Zhang, M. Seibert, and M. L. Ghirardi, J. Biol. Chem. 279:25711-25720, 2004). Previous efforts to study [FeFe] hydrogenase maturation in Escherichia coli by coexpression of C. reinhardtii HydEF and HydG and the HydA1 [FeFe] hydrogenase were hindered by instability of the hydEF and hydG expression clones. A more stable [FeFe] hydrogenase expression system has been achieved in E. coli by cloning and coexpression of hydE, hydF, and hydG from the bacterium Clostridium acetobutylicum. Coexpression of the C. acetobutylicum maturation proteins with various algal and bacterial [FeFe] hydrogenases in E. coli resulted in purified enzymes with specific activities that were similar to those of the enzymes purified from native sources. In the case of structurally complex [FeFe] hydrogenases, maturation of the catalytic sites could occur in the absence of an accessory iron-sulfur cluster domain. Initial investigations of the structure and function of the maturation proteins HydE, HydF, and HydG showed that the highly conserved radical-SAM domains of both HydE and HydG and the GTPase domain of HydF were essential for achieving biosynthesis of active [FeFe] hydrogenases. Together, these results demonstrate that the catalytic domain and a functionally complete set of Hyd maturation proteins are fundamental to achieving biosynthesis of catalytic [FeFe] hydrogenases.

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