Endoglucanase G from Fibrobacter succinogenes S85 belongs to a class of enzymes characterized by a basic C-terminal domain

1996 ◽  
Vol 42 (9) ◽  
pp. 934-943 ◽  
Author(s):  
Abiye H. Iyo ◽  
Cecil W. Forsberg
Keyword(s):  
Catalytic Domain ◽  
Specific Activity ◽  
Glutamate Synthase ◽  
Open Reading Frames ◽  
Crystalline Cellulose ◽  
Fibrobacter Succinogenes ◽  
Ph Optimum ◽  
E Coli ◽  
Terminal Domain ◽  
Orf2 Protein

A 3.6-kb fragment of the Fibrobacter succinogenes S85 DNA was sequenced and found to contain two open reading frames (ORFs) on the same strand separated by 242 nucleotide bases. The translated protein from ORF1 had a predicted mass of 52.3 kDa. In a region of 320 amino acid overlap, it shares a 35% identity with the b-chain of the glutamate synthase of Escherichia coli. The ORF2 protein encodes a 519 residue protein designated CelG. It consists of an ORF of 1557 bp, encoding a polypeptide of 54.5 kDa. The N-terminal region, which contains the catalytic domain, is linked to a C-terminal basic domain, which has a predicted isoelectric point of 10.8. The catalytic domain in endoglucanase G (CelG) is homologous to the family 5 (A) cellulases. The enzyme has an apparent mass of 55 kDa, a pH optimum of 5.5, and temperature optimum of 25 °C. It had a specific activity of 16.5 mmol∙min−1∙mg−1 on barley b-glucan and produced a mixture of cellooligosaccharides from the hydrolysis of acid swollen cellulose and cellooligosaccharides. Antiserum raised against the purified form of CelG in E. coli failed to react with proteins from the native organism when grown on either glucose or crystalline cellulose, but reverse transcription and polymerase chain reaction techniques using RNA from the native organism demonstrated that the celG gene was expressed constitutively. Its distribution amongst subspecies of Fibrobacter was restricted to F. succinogenes S85.Key words: basic terminal domain, Fibrobacter succinogenes, endoglucanase, nucleotide sequence.

Characteristics of a cluster of xylanase genes inFibrobacter succinogenesS85

2003 ◽  
Vol 49 (3) ◽  
pp. 171-180 ◽  
Author(s):  
Hyun S Jun ◽  
Jong K Ha ◽  
Laercio M Malburg, Jr. ◽  
Ann M Verrinder Gibbins ◽  
Cecil W Forsberg
Keyword(s):  
Catalytic Domain ◽  
Specific Activity ◽  
Glycosyl Hydrolase ◽  
Amino Acid Sequences ◽  
Pcr Analysis ◽  
Carbohydrate Binding ◽  
Fibrobacter Succinogenes ◽  
Rt Pcr ◽  
Ph Optimum ◽  
Oat Spelt

Xylanase genes xyn10D, xyn10E, and xyn10B, located sequentially on the Fibrobacter succinogenes S85 chromosome, were separately cloned and their properties characterized. Analysis of the sequences documented that xylanases Xyn10D, Xyn10E, and Xyn10B each consist of an N-terminal catalytic domain (glycosyl hydrolase family 10) and a C-terminal carbohydrate-binding module (CBM, family 6) connected by proline-rich linker sequences. The amino acid sequences exhibited similarities of between 53 and 60%. The xyn10D, xyn10E, and truncated xyn10BΔCBM were expressed in Escherichia coli and purified to homogeneity. The purified Xyn10D, Xyn10E, and Xyn10BΔCBM exhibited the same temperature optimum (40°C) and pH optimum (6.5) and the highest specific activity against arabinoxylan, oat spelt xylan, and birchwood xylan, respectively. Xyn10D exhibited an affinity for cellulose and xylan with 47 and 33% binding, respectively, while the truncated Xyn10DΔCBM did not bind to the substrates. The main hydrolysis products of the three xylanases acting on oat spelt xylan and arabinoxylan were xylose and xylobiose. RT-PCR analysis showed that the three genes were co-transcribed as a single transcript. Western immunoblot analysis revealed that the three xylanases were expressed at a very low level by F. succinogenes grown on either glucose or cellulose as the source of carbohydrate.Key words: Fibrobacter succinogenes S85, xylan, xylanase, clustered genes, RT-PCR.

scholarly journals Regulation of Escherichia coli RelA Requires Oligomerization of the C-Terminal Domain

2001 ◽  
Vol 183 (2) ◽  
pp. 570-579 ◽  
Author(s):  
Michal Gropp ◽  
Yael Strausz ◽  
Miriam Gross ◽  
Gad Glaser
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Catalytic Domain ◽  
Mutational Analysis ◽  
Amino Acid Starvation ◽  
E Coli ◽  
Terminal Domain ◽  
Negative Effect ◽  
And Control ◽  
Two Hybrid

ABSTRACT The E. coli RelA protein is a ribosome-dependent (p)ppGpp synthetase that is activated in response to amino acid starvation. RelA can be dissected both functionally and physically into two domains: The N-terminal domain (NTD) (amino acids [aa] 1 to 455) contains the catalytic domain of RelA, and the C-terminal domain (CTD) (aa 455 to 744) is involved in regulating RelA activity. We used mutational analysis to localize sites important for RelA activity and control in these two domains. We inserted two separate mutations into the NTD, which resulted in mutated RelA proteins that were impaired in their ability to synthesize (p)ppGpp. When we caused the CTD inrelA + cells to be overexpressed, (p)ppGpp accumulation during amino acid starvation was negatively affected. Mutational analysis showed that Cys-612, Asp-637, and Cys-638, found in a conserved amino acid sequence (aa 612 to 638), are essential for this negative effect of the CTD. When mutations corresponding to these residues were inserted into the full-length relA gene, the mutated RelA proteins were impaired in their regulation. In attempting to clarify the mechanism through which the CTD regulates RelA activity, we found no evidence for competition for ribosomal binding between the normal RelA and the overexpressed CTD. Results from CyaA complementation experiments of the bacterial two-hybrid system fusion plasmids (G. Karimova, J. Pidoux, A. Ullmann, and D. Ladant, Proc. Natl. Acad. Sci. USA 95:5752–5756, 1998) indicated that the CTD (aa 564 to 744) is involved in RelA-RelA interactions. Our findings support a model in which RelA activation is regulated by its oligomerization state.

Thermostable esterase estUT1 of bacteria Ureibacillus thermosphaericus: the influence of additional processed domain TrxA on enzyme properties

2019 ◽  
Vol 19 (5) ◽  
pp. 399-407
Author(s):  
K. N. Sorokina ◽  
Yu. V. Samoilova ◽  
V. N. Parmon
Keyword(s):  
Specific Activity ◽  
Enzyme Properties ◽  
Ph Optimum ◽  
T7 Promoter ◽  
Genetic Construct ◽  
E Coli ◽  
Additional Domain ◽  
Ureibacillus Thermosphaericus ◽  
The Stability ◽  
Dissolved Form

A gene of thermostable esterase of bacteria Ureibacillus thermosphaericus was expressed in strain E. coli BL21(DE3) comprised in domain TrxA-containing genetic construct pET32b-estUT1, under the control T7-promoter. The specific activity and relative thermostability of thus produced recombinant enzyme increased from 54.2 to 65.8 % (an hour incubation at 70 °C). The additional domain TrxA was discovered not to affect noticeably the pH optimum of the enzyme activity and its substrate specificity. In the absence of domain TrxA, the stability of estUT1 increased considerably in the presence of various chemicals including ethanol and methanol. The maximal catalytic activity (kcat/KM) of esterase equal to 280.0 s–1·mM–1 was observed in the absence of domain TrxA. Thus, introduction of an additional processed domain TrxA allows the enzyme to be secreted in the dissolved form but, on the other hand, the target protein becomes less thermostable.

scholarly journals Towards Designer Cellulosomes in Clostridia: Mannanase Enrichment of the Cellulosomes Produced by Clostridium cellulolyticum

2004 ◽  
Vol 186 (19) ◽  
pp. 6544-6552 ◽  
Author(s):  
Stéphanie Perret ◽  
Anne Bélaich ◽  
Henri-Pierre Fierobe ◽  
Jean-Pierre Bélaich ◽  
Chantal Tardif
Keyword(s):  
Guar Gum ◽  
Specific Activity ◽  
Full Length ◽  
Expression Vectors ◽  
Leader Peptide ◽  
Crystalline Cellulose ◽  
Glycosyl Hydrolases ◽  
Truncated Form ◽  
E Coli ◽  
Clostridium Cellulolyticum

ABSTRACT The man5K gene of Clostridium cellulolyticum was cloned and overexpressed in Escherichia coli. This gene encodes a 424-amino-acid preprotein composed of an N-terminal leader peptide, followed by a dockerin module and a C-terminal catalytic module belonging to family 5 of the glycosyl hydrolases. Mature Man5K displays 62% identity with ManA from Clostridium cellulovorans. Two forms of the protein were purified from E. coli; one form corresponds to the full-length enzyme (45 kDa), and a truncated form (39 kDa) lacks the N-terminal dockerin module. Both forms exhibit the same typical family 5 mannanase substrate preference; they are very active with the galactomannan locust bean gum, and the more galacto-substituted guar gum molecules are degraded less. The truncated form, however, displays fourfold-higher activity with galactomannans than the full-length enzyme. Man5K was successfully overproduced in C. cellulolyticum by using expression vectors. The trans-produced protein was found to be incorporated into the cellulosomes and became one of the major enzymatic components. Modified cellulosomes displayed 20-fold-higher specific activities than control fractions on galactomannan substrates, whereas the specific activity on crystalline cellulose was reduced by 20%. This work clearly showed that the composition of the cellulosomes is obviously regulated by the relative amounts of the enzymes produced and that this composition can be engineered in clostridia by structural gene cloning.

scholarly journals Purification, characterization, DNA sequence and cloning of a pimeloyl-CoA synthetase from Pseudomonas mendocina 35

1999 ◽  
Vol 340 (3) ◽  
pp. 793-801 ◽  
Author(s):  
Andrew BINIEDA ◽  
Martin FUHRMANN ◽  
Bruno LEHNER ◽  
Claudine REY-BERTHOD ◽  
Séverine FRUTIGER-HUGHES ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dna Sequence ◽  
Specific Activity ◽  
Amino Acid Sequences ◽  
Active Enzyme ◽  
Gene Probe ◽  
Pimelic Acid ◽  
Pseudomonas Mendocina ◽  
Ph Optimum ◽  
E Coli

A pimeloyl-CoA synthetase from Pseudomonas mendocina 35 was purified and characterized, the DNA sequence determined, and the gene cloned into Escherichia coli to yield an active enzyme. The purified enzyme had a pH optimum of ≈ 8.0, Km values of 0.49 mM for pimelic acid, 0.18 mM for CoA and 0.72 mM for ATP, a subunit Mr of ≈ 80000 as determined by SDS/PAGE, and was found to be a tetramer by gel-filtration chromatography. The specific activity of the purified enzyme was 77.3 units/mg of protein. The enzyme was not absolutely specific for pimelic acid. The relative activity for adipic acid (C6) was 72% and for azaleic acid (C9) was 18% of that for pimelic acid (C7). The N-terminal amino acid was blocked to amino acid sequencing, but controlled proteolysis resulted in three peptide fragments for which amino acid sequences were obtained. An oligonucleotide gene probe corresponding to one of the amino acid sequences was synthesized and used to isolate the gene (pauA, imelic cid-tilizing ) coding for pimeloyl-CoA synthetase. The pauA gene, which codes for a protein with a theoretical Mr of 74643, was then sequenced. The deduced amino acid sequence of the enzyme showed similarity to hypothetical proteins from Archaeoglobus fulgidus, Methanococcus jannaschii, Pyrococcus horikoshii, E. coli and Streptomyces coelicolor, and some limited similarity to microbial succinyl-CoA synthetases. The similarity with the protein from A. fulgidus was especially strong, thus indicating a function for this unidentified protein. The pauA gene was cloned into E. coli, where it was expressed and resulted in an active enzyme.

scholarly journals Cloning, expression in Komagataella phaffii, and biochemical characterization of recombinant sequence variants of Pseudomonas sp. S9 GDSL-esterase

2021 ◽  
Author(s):  
Monika Wicka-Grochocka ◽  
Hubert Cieśliński ◽  
Marta Wanarska
Keyword(s):  
Biochemical Characterization ◽  
Catalytic Domain ◽  
Specific Activity ◽  
Expression System ◽  
Single Domain ◽  
Pseudomonas Sp ◽  
E Coli ◽  
Komagataella Phaffii ◽  
Pichia Pastoris Yeast ◽  
Psychrotolerant Bacterium

Two recombinant Komagataella phaffii (formerly Pichia pastoris) yeast strains for production of two sequential variants of EstS9 esterase from psychrotolerant bacterium Pseudomonas sp. S9, i.e. αEstS9N (a two-domain enzyme consisting of a catalytic domain and an autotransporter domain) and αEstS9Δ (a single-domain esterase) were constructed. However, only one of recombinant K. phaffii strains, namely Komagataella phaffii X-33/pPICZαestS9Δ, allowed to successfully produce and secrete recombinant αEstS9Δ enzyme outside of the host cell. The purified αEstS9Δ esterase was active towards short-chain p-nitrophenyl esters (C2–C8), with optimal activity for the acetate (C2) ester. The single-domain αEstS9Δ esterase exhibits the highest activity at 60oC and pH 9.5. In addition, the enzyme retains 90% of its activity after 3 hour incubation at 70–90oC. What should be also noted is that αEstS9Δ esterase produced in the K. phaffii expression system has a much higher specific activity (0.069 U/mg of protein) than the recombinant EstS9Δ esterase produced in an E. coli expression system (0.0025 U/mg of protein) (Wicka et al., 2016, Acta Biochim Pol 63: 117–125. https://doi.org/10.18388/abp.2015_1074).

scholarly journals The purification and properties of the β-lactamase specified by the resistance factor R-1818 in Escherichia coli and Proteus mirabilis

1971 ◽  
Vol 123 (4) ◽  
pp. 493-500 ◽  
Author(s):  
J. W. Dale ◽  
J. T. Smith
Keyword(s):  
Escherichia Coli ◽  
Proteus Mirabilis ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Resistance Factor ◽  
Ph Optimum ◽  
E Coli ◽  
R Factor ◽  
Purification And Properties

1. The β-lactamase specified by the R-1818 resistance factor in Escherichia coli was purified 300-fold; the resulting preparation gave a single peak on Sephadex G-100 and a single band on polyacrylamide-gel electrophoresis. 2. The β-lactamase specified by the same R-factor in Proteus mirabilis was purified over 2000-fold, but was still far from pure. The specific activity of this preparation was one-fifth that of the purified enzyme from E. coli. 3. The two enzymes were shown to be identical as regards substrate specificity, pH optimum, Km values and molecular weight. 4. It is suggested that the low β-lactamase activity of extracts of P. mirabilis (R-1818), about 5% of that from E. coli (R-1818) in crude extracts, could be due to inefficient transcription of the R-factor DNA by Proteus RNA polymerase.

scholarly journals The non-catalytic C-terminal region of endoglucanase E from Clostridium thermocellum contains a cellulose-binding domain

1991 ◽  
Vol 273 (2) ◽  
pp. 289-293 ◽  
Author(s):  
A J Durrant ◽  
J Hall ◽  
G P Hazlewood ◽  
H J Gilbert
Keyword(s):  
Amino Acids ◽  
Clostridium Thermocellum ◽  
Catalytic Domain ◽  
Specific Activity ◽  
Binding Domain ◽  
Full Length ◽  
Crystalline Cellulose ◽  
Cellulose Binding Domain ◽  
Beta Glucan ◽  
Cellulose Binding

Mature endoglucanase E (EGE) from Clostridium thermocellum consists of 780 amino acid residues and has an Mr of 84,016. The N-terminal 334 amino acids comprise a functional catalytic domain. Full-length EGE bound to crystalline cellulose (Avicel) but not to xylan. Bound enzyme could be eluted with distilled water. The capacity of truncated derivatives of the enzyme to bind cellulose was investigated. EGE lacking 109 C-terminal residues (EGEd) or a derivative in which residues 367-432 of the mature form of the enzyme had been deleted (EGEb), bound to Avicel, whereas EGEa and EGEc, which lack 416 and 246 C-terminal residues respectively, did not. The specific activity of EGEa, consisting of the N-terminal 364 amino acids, was 4-fold higher than that of the full-length enzyme. The truncated derivative also exhibited lower affinity for the substrate beta-glucan than the full-length enzyme. It is concluded that EGE contains a cellulose-binding domain, located between residues 432 and 671, that is distinct from the active site. The role of this substrate-binding domain is discussed.

scholarly journals Modulation of Prohormone Convertase 1/3 Properties Using Site-Directed Mutagenesis

Endocrinology ◽  
2010 ◽  
Vol 151 (9) ◽  
pp. 4437-4445 ◽  
Author(s):  
Akihiko Ozawa ◽  
Juan R. Peinado ◽  
Iris Lindberg
Keyword(s):  
Catalytic Domain ◽  
Specific Activity ◽  
Site Directed Mutagenesis ◽  
Prohormone Convertase ◽  
Ph Optimum ◽  
Specific Domain ◽  
Molecular Determinant ◽  
Prohormone Convertase 1 ◽  
Domain Interactions ◽  
Endogenous Substrate

Prohormone convertase (PC)1/3 and PC2 cleave active peptide hormones and neuropeptides from precursor proteins. Compared with PC2, recombinant PC1/3 exhibits a very low specific activity against both small fluorogenic peptides and recombinant precursors, even though the catalytic domains in mouse PC1/3 and PC2 share 56% amino acid sequence identity. In this report, we have designed PC2-specific mutations into the catalytic domain of PC1/3 in order to investigate the molecular contributions of these sequences to PC1/3-specific properties. The exchange of residues RQG314 with the SY sequence present in the same location within PC2 paradoxically shifted the pH optimum of PC1/3 upward into the neutral range; other mutations in the catalytic domain had no effect. Although none of the full-length PC1/3 mutants examined exhibited increased specific activity, the 66-kDa form of the RQG314SY mutant was two to four times more active than the 66-kDa form of wild-type PC1/3. However, stable transfection of RQG314SY into PC12 cells did not result in greater activity against the endogenous substrate proneurotensin, implying unknown cellular controls of PC1/3 activity. Mutation of GIVTDA243–248 to QPFMTDI, a molecular determinant of 7B2 binding, resulted in increased zymogen expression but no propeptide cleavage or secretion, suggesting that this mutant is trapped in the endoplasmic reticulum due to an inability to cleave its own propeptide. We conclude that many convertase-specific properties are attributable less to convertase-specific catalytic cleft residues than to convertase-specific domain interactions.

scholarly journals An α-l-Arabinofuranosidase fromTrichoderma reesei Containing a Noncatalytic Xylan-Binding Domain

1999 ◽  
Vol 65 (9) ◽  
pp. 3964-3968 ◽  
Author(s):  
Masahiro Nogawa ◽  
Kenji Yatsui ◽  
Akiko Tomioka ◽  
Hirofumi Okada ◽  
Yasushi Morikawa
Keyword(s):  
Molecular Mass ◽  
Catalytic Domain ◽  
Specific Activity ◽  
Binding Domain ◽  
Amino Acid Sequences ◽  
Crystalline Cellulose ◽  
Terminal Amino Acid ◽  
Catalytic Constant ◽  
Terminal Amino ◽  
Oat Spelt

ABSTRACT l-Sorbose, an excellent cellulase and xylanase inducer from Trichoderma reesei PC-3-7, also induced α-l-arabinofuranosidase (α-AF) activity. An α-AF induced by l-sorbose was purified to homogeneity, and its molecular mass was revealed to be 35 kDa (AF35), which was not consistent with that of the previously reported α-AF. Another species, with a molecular mass of 53 kDa (AF53), which is identical to that of the reported α-AF, was obtained by a different purification procedure. Acid treatment of the ammonium sulfate-precipitated fraction at pH 3.0 in the purification steps or pepsin treatment of the purified AF53 reduced the molecular mass to 35 kDa. Both purified enzymes have the same enzymological properties, such as pH and temperature effects on activity and kinetic parameters forp-nitrophenyl-α-l-arabinofuranoside (pNPA). Moreover, the N-terminal amino acid sequences of these enzymes were identical with that of the reported α-AF. Therefore, it is obvious that AF35 results from the proteolytic cleavage of the C-terminal region of AF53. Although AF35 and AF53 showed the same catalytic constant with pNPA, the former showed drastically reduced specific activity against oat spelt xylan compared to the latter. Furthermore, AF53 was bound to xylan rather than to crystalline cellulose (Avicel), but AF35 could not be bound to any of the glycans. These results suggest that AF53 is a modular glycanase, which consists of an N-terminal catalytic domain and a C-terminal noncatalytic xylan-binding domain.

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