Pullulanase of Thermoanaerobacterium thermosulfurigenes EM1 (Clostridium thermosulfurogenes): molecular analysis of the gene, composite structure of the enzyme, and a common model for its attachment to the cell surface

Matuschek, Markus; Burchhardt, Gerhard; Sahm, Kerstin; Bahl, Hubert

doi:10.1128/jb.176.11.3295-3302.1994

Cited by 87 publications

(70 citation statements)

References 46 publications

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“…The SLH domain was postulated to be the determinant for interaction of the S-protein and other extracellular proteins with the underlying peptidoglycan layer (Matuschek et al, 1994). Another explanation is that the SLH domain of such extracellular enzymes interacts with (SLH) domains of the S-protein (Egelseer et al, 1995).…”

Section: Interaction Between S-proteins and Cell-wall Componentsmentioning

confidence: 99%

“…Although some particular functions could be assigned to specific S-proteins, such as barriers between the cell and the environment (Paula et al, 1988) and attachment structure for extracellular enzymes (Matuschek et al, 1994), no general function has been described which is found for all S-layers. For Archaebacteria, it has been reported that the S-layer plays a role in maintenance of the cell shape (Pum et al, 1991).…”

Section: Introductionmentioning

confidence: 99%

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Expression, secretion and antigenic variation of bacterial S‐layer proteins

Boot

Pouwels

1996

Molecular Microbiology

120

108

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SummaryThe function of the S-layer, a regularly arranged structure on the outside of numerous bacteria, appears to be different for bacteria living in different environments. Almost no similarity exists between the primary sequences of S-proteins, although their amino acid composition is comparable. S-protein production is directed by single or multiple promoters in front of the S-protein gene, yielding stable mRNAs. Most bacteria secrete S-proteins via the general secretory pathway (GSP). Translocation of S-protein across the outer membrane of Gram-negative bacteria sometimes occurs by S-protein-specific branches of the GSP. O-polysaccharide side-chains of the lipopolysaccharide component of the cell wall of Gramnegative bacteria appear to function as receptors for attachment of the S-layer. Silent S-protein genes have been found in Campylo-bacter fetus and Lactobacillus acidophilus. These silent genes are placed in the expression site in a fraction of the bacterial population via inversion of a chromosomal segment.

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Section: Interaction Between S-proteins and Cell-wall Componentsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Expression, secretion and antigenic variation of bacterial S‐layer proteins

Boot

Pouwels

1996

Molecular Microbiology

120

108

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“…8, part of the sequence of Amy is aligned with that of its three closests relatives [Tts and Tet, standing for the (amy1o)pullulanases from 7: thermosulfurigenes [26] and 7: ethanolicus [27], respectively, which are almost identical to the a-amylase pullulanase from 7: thermohydrosulfuricus [28], and Neo, the neopullulanase from B. stearothermophilus [50]]. In the aligned regions, Tts, Tet and Neo show 63, 60 and 56% sequence similarity with Amy, in which similarity is defined as the sum of the identical plus the conservatively exchanged residues (see the legend to Fig.…”

Section: Sequence Alignmentmentioning

confidence: 99%

“…Recently, Koivola et al [25] published the nucleotide sequence of this gene. Unexpectedly, its product is predicted to consist of as many as 1301 amino acid residues and to be closely related to a group of enzymes referred to as pullulanases, amylo-pullulanases, or a-amylases-pullulanases from Therrnoanaerobacterium thermosulfurigenes (formerly Clostridium thermosuljiurogenes) EM1 [26], Thermoanaerobacterium ethanolicus (formerly Clostridium thermohydosulfuricum) 39E [27] and Thermoanaerobacter (formerly Clostridium) thermohydrosulfuricum [28]. Koivola et al [25] did not isolate the A. acidocaldurius ATCC 27009 enzyme, but a zymogram shows that the main starch-degrading protein has an apparent molecular mass of 160000Da.…”

mentioning

confidence: 99%

Purification, Properties and Structural Aspects of a Thermoacidophilic α‐Amylase from Alicyclobacillus Acidocaldarius Atcc 27009

Schwermann

Pfau

Liliensiek

et al. 1994

European Journal of Biochemistry

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The a-amylase from the thermoacidophilic eubacterium Alicyclobacillus (Bacillus) acidocaldarius strain ATCC 27009 was studied as an example of an acidophilic protein. The enzyme was purified from the culture fluid. On an SDS/polyacrylamide gel, the protein exhibited an apparent molecular mass of 160 kDa, which is approximately 15% higher than that predicted from the nucleotide sequence. The difference is due to the enzyme being a glycoprotein. Deglycosylation or synthesis of the enzyme in Escherichia coli gave a product with the mass expected for the mature protein.The amylase hydrolyzed starch at random and from the inside, and its main hydrolysis products were maltotriose and maltose. It also formed glucose from starch (by hydrolysing the intermediate product maltotetraose to glucose and maltotriose) and exhibited some pullulanase activity. The pH and temperature optima were pH 3 and 75 "C, respectively, characterizing the enzyme as being thermoacidophilic. Alignment of the sequence of the enzyme with that of its closests neutrophilic relatives and with that of a-1,4 or a-1,6 glycosidic-bond hydrolyzing enzymes of known threedimensional structure showed that the acidophilic a-amylase contains approximately 30% less charged residues than do its closests relatives, that these residues are replaced by neutral polar residues, and that hot spots for these exchanges are likely to be located at the surface of the protein.Literature data show that similar effects are observed in three other acidophilic proteins. It is proposed that these proteins have adapted to the acidic environment by reducing the density of both positive and negative charges at their surface, that this effect circumvents electrostatic repulsion of charged groups at low pH, and thereby contributes to the acidostability of these proteins.Enzymes optimally active under extreme conditions are both of biotechnological importance and of scientific interest (for reviews see [I-31). Much work has been devoted to the mechanism of thermostability. However, its outcome is complex. Theoretical considerations suggest that the exchange of a single amino acid residue in an enzyme can increase its temperature optimum by more than 10°C [3]. Studies on forced molecular evolution have provided support for this notion. In such thermo-drill experiments, a gene encoding a mesophilic enzyme is introduced into a thermophilic organism, and by gradually increasing the growth tem-Correspondence to E. P. Bakker, Abteilung Mikrobiologie,

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“…Clostridium thermocellum, a thermophilic, anaerobic bacterium, is best known for producing highly active multienzyme cellulase complexes termed cellulosomes polysaccharides (Lupas et al, 1994;Matuschek et al, 1994). SLH domains appear to mediate attachment of proteins to the cell wall (Lemaire et al, 1995 ;Olabarria et al, 1996) as well as interactions with other SLH domains (Lemaire et al, 1995).…”

Section: Introductionmentioning

confidence: 99%

Identification of a region responsible for binding to the cell wall within the S-layer protein of Clostridium thermocellum

et al. 1998

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The protomer forming the Slayer of CIostridium thermocellum was identified as a 140 kDa protein which was non-covalently bound to the cell wall. Cloning and sequencing of the corresponding gene revealed an open reading frame of 3108 nucleotides encoding a polypeptide of 1036 amino acids, termed SlpA. The amino acid composition of SlpA matches the composition of a previously described exocellular glycoprotein. SlpA shared extensive similarity with the Slayer protein of Bacillus sphaericus and with the outer wall protein of Bacillus brewis. In addition, the amino-terminal region of SlpA contained a segment presenting similarities with segments termed SLH (Slayer homologous), which are found in several bacterial exoproteins. A polypeptide of 209 residues comprising this segment was shown to bind to cell walls extracted from C. thermoeellum cells.

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Pullulanase of Thermoanaerobacterium thermosulfurigenes EM1 (Clostridium thermosulfurogenes): molecular analysis of the gene, composite structure of the enzyme, and a common model for its attachment to the cell surface

Abstract: The complete pullulanase gene (amyB) from Thermoanaerobacterium thermosulfurigenes EM1 was cloned in Escherichia coli, and the nucleotide sequence was determined. The

Cited by 87 publications

References 46 publications

Expression, secretion and antigenic variation of bacterial S‐layer proteins

Expression, secretion and antigenic variation of bacterial S‐layer proteins

Purification, Properties and Structural Aspects of a Thermoacidophilic α‐Amylase from Alicyclobacillus Acidocaldarius Atcc 27009

Identification of a region responsible for binding to the cell wall within the S-layer protein of Clostridium thermocellum

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