Two genes encoding an endoglucanase and a cellulose-binding protein are clustered and co-regulated by a TTA codon in Streptomyces halstedii JM8

Streptomyces halstediiJM8 Cel2 is an endoglucanase of 28 kDa that is first produced as a protein of 42 kDa (p42) and is later processed at its C-terminus. Cel2 displays optimal activity towards CM-cellulose at pH 6 and 50 °C and shows no activity against crystalline cellulose or xylan. The N-terminus of p42 shares similarity with cellulases included in family 12 of the β-glycanases and the C-terminus shares similarity with bacterial cellulose-binding domains included in family II. This latter domain enables the precursor to bind so tightly to Avicel that it can only be eluted by boiling in 10% (w/v) SDS. Another open reading frame (ORF) situated 216 bp downstream from the p42 ORF encodes a protein of 40 kDa (p40) that does not have any clear hydrolytic activity against cellulosic or xylanosic compounds, but shows high affinity for Avicel (crystalline cellulose). The p40 protein is processed in old cultures to give a protein of 35 kDa that does not bind to Avicel. Translation of both ORFs is impaired in Streptomyces coelicolor bldA mutants, suggesting that a TTA codon situated at the fourth position of the first ORF is responsible for this regulation. S1 nuclease protection experiments demonstrate that both ORFs are co-transcribed.

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Xylanase B and an arabinofuranosidase from Pseudomonas fluorescens subsp. cellulosa contain identical cellulose-binding domains and are encoded by adjacent genes

Biochemical Journal ◽

10.1042/bj2720369 ◽

1990 ◽

Vol 272 (2) ◽

pp. 369-376 ◽

Cited By ~ 102

Author(s):

L E Kellett ◽

D M Poole ◽

L M Ferreira ◽

A J Durrant ◽

G P Hazlewood ◽

...

Keyword(s):

Alkaline Phosphatase ◽

Pseudomonas Fluorescens ◽

Crystalline Cellulose ◽

Amino Acid Residues ◽

Gene Encoding ◽

Reading Frame ◽

Conserved Sequence ◽

Binding Domains ◽

Cellulose Binding ◽

Derivatives Of

The complete nucleotide sequence of the Pseudomonas fluorescens subsp. cellulosa xynB gene, encoding an endo-beta-1,4-xylanase (xylanase B; XYLB) has been determined. The structural gene consists of an open reading frame (ORF) of 1775 bp coding for a protein of Mr 61,000. A second ORF (xynC) of 1712 bp, which starts 148 bp downstream of xynB, encodes a protein, designated xylanase C (XYLC), of Mr 59,000. XYLB hydrolyses oat spelt xylan to xylobiose and xylose, whereas XYLC releases only arabinose from the same substrate. Thus XYLB is a typical xylanase and XYLC is an arabinofuranosidase. Both enzymes bind to crystalline cellulose (Avicel), but not to xylan. The nucleotide sequences between residues 114 and 931 of xynB and xynC were identical, as were amino acid residues 39-311 of XYLB and XYLC. This conserved sequence is reiterated elsewhere in the P. fluorescens subsp. cellulosa genome. Truncated derivatives of XYLB and XYLC, in which the conserved sequence had been deleted, retained catalytic activity, but did not exhibit cellulose binding. A hybrid gene in which the 5′ end of xynC, encoding residues 1-110 of XYLC, was fused to the Escherichia coli pho A' gene (encodes mature alkaline phosphatase) directed the synthesis of a fusion protein which exhibited alkaline phosphatase activity and bound to cellulose.

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The cellodextrinase from Pseudomonas fluorescens subsp. cellulosa consists of multiple functional domains

Biochemical Journal ◽

10.1042/bj2790793 ◽

1991 ◽

Vol 279 (3) ◽

pp. 793-799 ◽

Cited By ~ 18

Author(s):

L M A Ferreira ◽

G P Hazlewood ◽

P J Barker ◽

H J Gilbert

Keyword(s):

Pseudomonas Fluorescens ◽

Catalytic Domain ◽

Genomic Library ◽

Terminal Region ◽

Crystalline Cellulose ◽

Nucleotide Sequencing ◽

Reading Frame ◽

Strong Homology ◽

Cellulose Binding

A genomic library of Pseudomonas fluorescens subsp. cellulosa DNA was constructed in pUC18 and Escherichia coli recombinants expressing 4-methylumbelliferyl beta-D-cellobioside-hydrolysing activity (MUCase) were isolated. Enzyme produced by MUCase-positive clones did not hydrolyse either cellobiose or cellotriose but converted cellotetraose into cellobiose and cleaved cellopentaose and cellohexaose, producing a mixture of cellobiose and cellotriose. There was no activity against CM-cellulose, insoluble cellulose or xylan. On this basis, the enzyme is identified as an endo-acting cellodextrinase and is designated cellodextrinase C (CELC). Nucleotide sequencing of the gene (celC) which directs the synthesis of CELC revealed an open reading frame of 2153 bp, encoding a protein of Mr 80,189. The deduced primary sequence of CELC was confirmed by the Mr of purified CELC (77,000) and by the experimentally determined N-terminus of the enzyme which was identical with residues 38-47 of the translated sequence. The N-terminal region of CELC showed strong homology with endoglucanase, xylanases and an arabinofuranosidase of Ps. fluorescens subsp. cellulosa; homologous sequences included highly conserved serine-rich regions. Full-length CELC bound tightly to crystalline cellulose. Truncated forms of celC from which the DNA sequence encoding the conserved domain had been deleted, directed the synthesis of a functional cellodextrinase that did not bind to crystalline cellulose. This is consistent with the N-terminal region of CELC comprising a non-catalytic cellulose-binding domain which is distinct from the catalytic domain. The role of the cellulose-binding region is discussed.

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Characterization of hybrid proteins consisting of the catalytic domains of Clostridium and Ruminococcus endoglucanases, fused to Pseudomonas non-catalytic cellulose-binding domains

Biochemical Journal ◽

10.1042/bj2790787 ◽

1991 ◽

Vol 279 (3) ◽

pp. 787-792 ◽

Cited By ~ 22

Author(s):

D M Poole ◽

A J Durrant ◽

G P Hazlewood ◽

H J Gilbert

Keyword(s):

Catalytic Domain ◽

Binding Capacity ◽

Binding Domains ◽

Hybrid Proteins ◽

C Terminus ◽

N Terminus ◽

Fusion Enzymes ◽

Cellulose Binding

The N-terminal 160 or 267 residues of xylanase A from Pseudomonas fluorescens subsp. cellulosa, containing a non-catalytic cellulose-binding domain (CBD), were fused to the N-terminus of the catalytic domain of endoglucanase E (EGE') from Clostridium thermocellum. A further hybrid enzyme was constructed consisting of the 347 N-terminal residues of xylanase C (XYLC) from P. fluorescens subsp. cellulosa, which also constitutes a CBD, fused to the N-terminus of endoglucanase A (EGA) from Ruminococcus albus. The three hybrid enzymes bound to insoluble cellulose, and could be eluted such that cellulose-binding capacity and catalytic activity were retained. The catalytic properties of the fusion enzymes were similar to EGE' and EGA respectively. Residues 37-347 and 34-347 of XYLC were fused to the C-terminus of EGE' and the 10 amino acids encoded by the multiple cloning sequence of pMTL22p respectively. The two hybrid proteins did not bind cellulose, although residues 39-139 of XYLC were shown previously to constitute a functional CBD. The putative role of the P. fluorescens subsp. cellulosa CBD in cellulase action is discussed.

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The bldC Developmental Locus of Streptomyces coelicolor Encodes a Member of a Family of Small DNA-Binding Proteins Related to the DNA-Binding Domains of the MerR Family

Journal of Bacteriology ◽

10.1128/jb.187.2.716-728.2005 ◽

2005 ◽

Vol 187 (2) ◽

pp. 716-728 ◽

Cited By ~ 24

Author(s):

Alison C. Hunt ◽

Luis Servín-González ◽

Gabriella H. Kelemen ◽

Mark J. Buttner

Keyword(s):

Dna Binding ◽

Binding Proteins ◽

Antibiotic Production ◽

Streptomyces Coelicolor ◽

Aerial Mycelium ◽

Dna Binding Proteins ◽

S1 Nuclease ◽

Dna Binding Domains ◽

Binding Domains ◽

Wide Range

ABSTRACT The bldC locus, required for formation of aerial hyphae in Streptomyces coelicolor, was localized by map-based cloning to the overlap between cosmids D17 and D25 of a minimal ordered library. Subcloning and sequencing showed that bldC encodes a member of a previously unrecognized family of small (58- to 78-residue) DNA-binding proteins, related to the DNA-binding domains of the MerR family of transcriptional activators. BldC family members are found in a wide range of gram-positive and gram-negative bacteria. Constructed ΔbldC mutants were defective in differentiation and antibiotic production. They failed to form an aerial mycelium on minimal medium and showed severe delays in aerial mycelium formation on rich medium. In addition, they failed to produce the polyketide antibiotic actinorhodin, and bldC was shown to be required for normal and sustained transcription of the pathway-specific activator gene actII-orf4. Although ΔbldC mutants produced the tripyrrole antibiotic undecylprodigiosin, transcripts of the pathway-specific activator gene (redD) were reduced to almost undetectable levels after 48 h in the bldC mutant, in contrast to the bldC + parent strain in which redD transcription continued during aerial mycelium formation and sporulation. This suggests that bldC may be required for maintenance of redD transcription during differentiation. bldC is expressed from a single promoter. S1 nuclease protection assays and immunoblotting showed that bldC is constitutively expressed and that transcription of bldC does not depend on any of the other known bld genes. The bldC18 mutation that originally defined the locus causes a Y49C substitution that results in instability of the protein.

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Spatial separation of protein domains is not necessary for catalytic activity or substrate binding in a xylanase

Biochemical Journal ◽

10.1042/bj2690261 ◽

1990 ◽

Vol 269 (1) ◽

pp. 261-264 ◽

Cited By ~ 55

Author(s):

L M A Ferreira ◽

A J Durrant ◽

J Hall ◽

G P Hazlewood ◽

H J Gilbert

Keyword(s):

Catalytic Activity ◽

Specific Activity ◽

Binding Capacity ◽

Spatial Separation ◽

Crystalline Cellulose ◽

Conserved Sequence ◽

Binding Domains ◽

Catalytically Active ◽

Cellulose Binding ◽

Derivatives Of

Xylanase A (XYLA) from Pseudomonas fluorescens subspecies cellulosa shows sequence conservation with two endoglucanases from the same organism. The conserved sequence in XYLA, consisting of the N-terminal 234 residues, is not essential for catalytic activity. Full-length XYLA and a fusion enzyme, consisting of the N-terminal 100 residues of XYLA linked to mature alkaline phosphatase, bound tightly to crystalline cellulose (Avicel), but not to xylan. The capacity of truncated derivatives of the xylanase to bind polysaccharides was investigated. XYLA lacking the first 13 N-terminal amino acids did not bind to cellulose. However, a catalytically active XYLA derivative (XYLA′), in which residues 100-234 were deleted, bound tightly to Avicel. Substrate specificity, cellulose-binding capacity, specific activity and Km for xylan hydrolysis were evaluated for each of the xylanases. No differences in any of these parameters were detected for the two enzymes. It is concluded that XYLA contains a cellulose-binding domain consisting of the N-terminal 100 residues which is distinct from the active site. Spatial separation of the catalytic and cellulose-binding domains is not essential for the enzyme to function normally.

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Expression and characterization of a cutinase (AnCUT2) from Aspergillus niger

Open Life Sciences ◽

10.1515/biol-2016-0004 ◽

2016 ◽

Vol 11 (1) ◽

pp. 29-38 ◽

Cited By ~ 2

Author(s):

Khadijah Ahmed Al-Tammar ◽

Othman Omar ◽

Abdul Munir Abdul Murad ◽

Farah Diba Abu Bakar

Keyword(s):

Aspergillus Niger ◽

Hydrolytic Activity ◽

Water Soluble ◽

Synthetic Fiber ◽

Amino Acid Residues ◽

Efficient Catalyst ◽

Reading Frame ◽

Synthetic Fibers ◽

Genes Encoding ◽

Bacteria And Fungi

AbstractCutin hydrolase (EC 3.1.1.74), an extracellular polyesterase found in pollens, bacteria and fungi, is an efficient catalyst that exhibits hydrolytic activity on a variety of water-soluble esters, synthetic fibers, plastics and triglycerides. Thus, cutinase can be used in various applications such as ester synthesis, bio-scouring, food and detergent industries. Ancut2 is one of five genes encoding cutinases present in the Aspergillus niger ATCC 10574 genome. The cDNA of Ancut2 comprising of an open reading frame of 816 bp encoding a protein of 271 amino acid residues, was isolated and expressed in Pichia pastoris. The partially purified recombinant cutinase exhibited a molecular mass of approximately 40 kDa. The enzyme showed highest activity at 40°C with a preference for acidic pH (5.0-6.0). AnCUT2 showed hydrolytic activity towards various p-nitrophenyl esters with preference towards shorter chain esters such as p-nitrophenyl butyrate (C4). Scanning Electron Microscopy demonstrated that AnCUT2 was capable of modifying surfaces of synthetic polycaprolactone and polyethylene terephthalate plastics. The properties of this enzyme suggest that it may be applied in synthetic fiber modification and fruit processing industries.

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A Novel Cell Surface-Anchored Cellulose-Binding Protein Encoded by the sca Gene Cluster of Ruminococcus flavefaciens

Journal of Bacteriology ◽

10.1128/jb.00143-07 ◽

2007 ◽

Vol 189 (13) ◽

pp. 4774-4783 ◽

Cited By ~ 36

Author(s):

Marco T. Rincon ◽

Tadej Cepeljnik ◽

Jennifer C. Martin ◽

Yoav Barak ◽

Raphael Lamed ◽

...

Keyword(s):

Cell Surface ◽

Binding Protein ◽

Scaffolding Proteins ◽

Ruminococcus Flavefaciens ◽

Reading Frame ◽

C Terminus ◽

A Cell ◽

Mutant Derivative ◽

Cellulose Binding ◽

Recombinant Fragments

ABSTRACT Ruminococcus flavefaciens produces a cellulosomal enzyme complex, based on the structural proteins ScaA, -B, and -C, that was recently shown to attach to the bacterial cell surface via the wall-anchored protein ScaE. ScaA, -B, -C, and -E are all cohesin-bearing proteins encoded by linked genes in the sca cluster. The product of an unknown open reading frame within the sca cluster, herein designated CttA, is similar in sequence at its C terminus to the corresponding region of ScaB, which contains an X module together with a dockerin sequence. The ScaB-XDoc dyad was shown previously to interact tenaciously with the cohesin of ScaE. Likewise, avid binding was confirmed between purified recombinant fragments of the CttA-XDoc dyad and the ScaE cohesin. In addition, the N-terminal regions of CttA were shown to bind to cellulose, thus suggesting that CttA is a cell wall-anchored, cellulose-binding protein. Proteomic analysis showed that the native CttA protein (∼130 kDa) corresponds to one of the three most abundant polypeptides binding tightly to insoluble cellulose in cellulose-grown R. flavefaciens 17 cultures. Interestingly, this protein was also detected among cellulose-bound proteins in the related strain R. flavefaciens 007C but not in a mutant derivative, 007S, that was previously shown to have lost the ability to grow on dewaxed cotton fibers. In R. flavefaciens, the presence of CttA on the cell surface is likely to provide an important mechanism for substrate binding, perhaps compensating for the absence of an identified cellulose-binding module in the major cellulosomal scaffolding proteins of this species.

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Sterol 14α-demethylase activity in Streptomyces coelicolor A3(2) is associated with an unusual member of the CYP51 gene family

Biochemical Journal ◽

10.1042/bj20011380 ◽

2002 ◽

Vol 364 (2) ◽

pp. 555-562 ◽

Cited By ~ 21

Author(s):

David C. LAMB ◽

Kay FOWLER ◽

Tobias KIESER ◽

Nigel MANNING ◽

Larissa M. PODUST ◽

...

Keyword(s):

Streptomyces Coelicolor ◽

Open Reading Frame ◽

Reading Frame ◽

E Coli ◽

Transposon Insertion ◽

Cell Extracts ◽

C Terminus ◽

Demethylase Activity ◽

First Time

The annotation of the genome sequence of Streptomyces coelicolor A3(2) revealed a cytochrome P450 (CYP) resembling various sterol 14α-demethylases (CYP51). The putative CYP open reading frame (SC7E4.20) was cloned with a tetrahistidine tag appended to the C-terminus and expressed in Escherichia coli. Protein purified to electrophoretic homogeneity was observed to bind the 14-methylated sterols lanosterol and 24-methylene-24,25-dihydrolanosterol (24-MDL). Reconstitution experiments with E. coli reductase partners confirmed activity in 14α-demethylation for 24-MDL, but not lanosterol. An S. coelicolor A3(2) mutant containing a transposon insertion in the CYP51 gene, which will abolish synthesis of the functional haemoprotein, was isolated as a viable strain, the first time a CYP51 has been identified as non-essential. The role of this CYP in bacteria is intriguing. No sterol product was detected in non-saponifiable cell extracts of the parent S. coelicolor A3(2) strain or of the mutant. S. coelicolor A3(2) CYP51 contains very few of the conserved CYP51 residues and, even though it can catalyse 14α-demethylation, it probably has another function in Streptomyces. We propose that it is a member of a new CYP51 subfamily.

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Roles of the Catalytic Domain and Two Cellulose Binding Domains of Thermomonospora fusca E4 in Cellulose Hydrolysis

Journal of Bacteriology ◽

10.1128/jb.180.7.1709-1714.1998 ◽

1998 ◽

Vol 180 (7) ◽

pp. 1709-1714 ◽

Cited By ~ 133

Author(s):

Diana Irwin ◽

Dong-Hoon Shin ◽

Sheng Zhang ◽

Brian K. Barr ◽

Joshua Sakon ◽

...

Keyword(s):

Filter Paper ◽

Recombinant Dna ◽

Catalytic Domain ◽

Cellulose Hydrolysis ◽

Hydrolytic Activity ◽

Thermomonospora Fusca ◽

Binding Domains ◽

The Family ◽

Recombinant Dna Techniques ◽

Cellulose Binding

ABSTRACT Thermomonospora fusca E4 is an unusual 90.4-kDa endocellulase comprised of a catalytic domain (CD), an internal family IIIc cellulose binding domain (CBD), a fibronectinlike domain, and a family II CBD. Constructs containing the CD alone (E4-51), the CD plus the family IIIc CBD (E4-68), and the CD plus the fibronectinlike domain plus the family II CBD (E4-74) were made by using recombinant DNA techniques. The activities of each purified protein on bacterial microcrystalline cellulose (BMCC), filter paper, swollen cellulose, and carboxymethyl cellulose were measured. Only the whole enzyme, E4-90, could reach the target digestion of 4.5% on filter paper. Removal of the internal family IIIc CBD (E4-51 and E4-74) decreased activity markedly on every substrate. E4-74 did bind to BMCC but had almost no hydrolytic activity, while E4-68 retained 32% of the activity on BMCC even though it did not bind. A low-activity mutant of one of the catalytic bases, E4-68 (Asp55Cys), did bind to BMCC, although E4-51 (Asp55Cys) did not. The ratios of soluble to insoluble reducing sugar produced after filter paper hydrolysis by E4-90, E4-68, E4-74, and E4-51 were 6.9, 3.5, 1.3, and 0.6, respectively, indicating that the family IIIc CBD is important for E4 processivity.

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