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Previous Article | Next Article 
Journal of Bacteriology, May 1999, p. 3284-3287, Vol. 181, No. 10
Unité de Physicochimie et
Biotechnologie des Polymères,
Received 28 December 1998/Accepted 19 March 1999
The gene encoding a hyperthermostable type II pullulanase produced
by Thermococcus hydrothermalis (Th-Apu) has been isolated. Analysis of a total of 5.2 kb of genomic DNA has revealed the presence
of three open reading frames, one of which (apuA) encodes the pullulanase. This enzyme is composed of 1,339 amino acid residues and exhibits a multidomain structure. In addition to a typical N-terminal signal peptide, Th-Apu possesses a catalytic domain, a
domain bearing S-layer homology-like motifs, a Thr-rich region, and a
potential C-terminal transmembrane domain. The presence of these
noncatalytic domains suggests that Th-Apu may be anchored to the cell
surface and be O glycosylated.
Pullulanases (EC 3.2.1.41) cleave
the Previously, we have characterized the amylolytic activities of T. hydrothermalis and have shown that this archaebacterium produces
several amylolytic enzymes, including at least one pullulanase (Th-Apu)
(13, 19). Purification and characterization of this enzyme
have revealed that it is highly thermostable and is capable of
hydrolyzing the Isolation and characterization of the Th-Apu gene and its
surrounding DNA environment.
In order to isolate the gene encoding
Th-Apu, we first performed N-terminal microsequencing of the native
protein purified from the medium of a T. hydrothermalis
AL662 culture. This yielded a sequence of 11 amino acid residues
(AEPKPLNVIIV) which was compared to the N-terminal sequence of the
mature pullulanase from P. furiosus (Pf-Apu)
(12). The high degree of similarity between these two small
regions (9 of 11 amino acid residues are identical) indicated that the
two proteins might share a high degree of overall homology. Therefore,
using the primary sequence of Pf-Apu, three degenerate oligonucleotides
were designed for use in standard PCRs using T. hydrothermalis AL662 genomic DNA as the template. In this way, specific sequences were amplified and then sequenced, thus providing the necessary sequence information to perform genome crawling in the
upstream and downstream directions. Likewise, a 5,200-bp sequence was
generated which contains the entire Th-Apu gene (designated apuA) flanked by two partial ORFs. Translation of the
partial ORFs generated two polypeptides of 118 and 261 amino acids,
respectively. The first of these proteins is highly similar (81%
identity) to the C-terminal part of the hypothetical MalG-like protein
from P. furiosus (10) and indeed displays
identity with several known MalG proteins, including that of
Escherichia coli (41%) (9). Similarly, the
second polypeptide exhibits strong identity with several MalK proteins,
including those of E. coli (57% identity) (14),
Salmonella typhimurium (56% identity), and
Enterobacter aerogenes (57% identity) (8),
leading us to the conclusion that this protein may be a MalK homologue.
The apparently promoterless apuA gene, surrounded by
these mal-like genes, is composed of 4,011 bp, 2,562 bp of
which show some similarity (~40%) to the sequence encoding Pf-Apu.
However, the P. furiosus and T. hydrothermalis sequences diverge after this point. Whereas
the Pf-Apu ORF ends at this position with a stop codon, the T. hydrothermalis ORF has an additional 1,449 bp before the stop
codon (TGA) is found.
Primary structure analysis of Th-Apu.
Translation of
apuA revealed a polypeptide sequence comprised of 1,339 amino acids which are arranged into several well-defined domains (Fig.
1). Like Pf-Apu, Th-Apu possesses an
N-terminal sequence (amino acids 2 to 27) which bears the
characteristics of a signal peptide (33). The ensuing
sequence (PUL), composed of 827 amino acids, presents a high degree of
sequence identity (79%) with Pf-Apu and, as such, probably represents
the catalytic domain. Beyond this point, three other domain types,
absent in Pf-Apu, can be distinguished. The first of these consists of
two almost identical sequence repeats (R1 and R2), each of which
possesses a repeated motif which, when compared to the PROSITE
database, shows similarity to the S-layer homology (SLH) signature.
Alignment of these repeated motifs with the prealigned sequences of
ProDom domain 1624 (7) confirmed this finding while
indicating that there is no complete consensus between the Th-Apu
motifs (Fig. 2). Indeed, the latter part
of the SLH consensus motif, defined as ILLA TS R
ASQ EDQ, where consensus amino acid residues are in
boldface (24) is absent in the first SLH motif of both
Th-Apu S-layer motif-bearing domains (SLD1 and SLD2), while being
barely distinguishable in the second SLH motif of each of these
domains. Although this sequence appears to be a well-conserved element
of the SLH motif, its absence does not necessarily indicate that the
Th-Apu-derived domains are not SLDs, since a large sequence diversity
exists, even among the known eubacterial SLH motifs. Indeed, a similar,
incomplete SLH motif in the SlpA protein of Clostridium
thermocellum has recently been described (21).
Furthermore, apart from this apparent divergence from the canonical
sequence, the alignment revealed that the thermococcal domains possess
most of the other pertinent features of a eubacterial SLD. In contrast,
comparison of the SLH motifs of Th-Apu with those of several proteins
from methanogenic archaebacteria (6, 28, 29) failed to
reveal anything more than superficial similarity.
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The Type II Pullulanase of Thermococcus
hydrothermalis: Molecular Characterization of the Gene and
Expression of the Catalytic Domain
ABSTRACT
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-1,6 glucosidic bonds in pullulan (2) and can be
classified according to the additional ability (type II) or inability
(type I) to degrade the -1,4 glucosidic bonds of other
polysaccharides (1). In recent years, a considerable number
of type II pullulanases (also termed amylopullulanases) have been
isolated from a wide variety of microorganisms, particularly thermophilic ones, since scientific interest in this class of enzymes
is motivated by industrial applications (11, 18, 23, 25, 26,
39). To date, four members of the archaeal order Thermococcales, Pyrococcus furiosus,
Thermococcus litoralis (3), P. woesei
(35), and T. celer (4), have been
described as pullulanase producers. In each case, the pullulanase
activity was described as type II and was localized in the culture
medium, indicating that these enzymes are secreted. The enzymes from
P. furiosus and T. litoralis have been described
as glycosylated and may be present in multiple forms. In addition, the
production of these enzymes appears to be inducible, with
malto-oligosaccharides being the principal inducers. Among these
enzymes, only those of P. furiosus and P. woesei
have been studied at the genetic level. In both cases, a gene encoding
a type II pullulanase has been isolated, cloned, and characterized,
although the actual nucleotide sequence of the P. woesei
type II pullulanase is not available for analysis.
-1,6 glucosidic bonds of pullulan, producing maltotriose, and both the -1,6 and -1,4 glucosidic bonds of starch, producing oligosaccharides (degree of polymerization, as low as
4) (12). In this work, we report the isolation and characterization of the gene encoding Th-Apu, as well as two other partial open reading frames (ORFs) which encode mal-like
operon elements.
View larger version (29K):
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FIG. 1.
Multidomain structure of Th-Apu and comparison of this
protein to Pf-Apu. R1 and R2 are two homologous, 230-amino-acid (aa)
residue repeats. SLD1 and SLD2 (checkered) are SLH motif-bearing
domains (containing approximately two and a half SLH motifs per
domain). SP is a signal peptide, PET is a Thr-rich domain, and TM is a
hydrophobic C-terminal domain.
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FIG. 2.
Alignment of the two Th-APu SLDs with a variety of
eubacterial SLDs. The alignment is based on ProDom domain 1624 (INRA,
Toulouse, France), which was constructed from 15 eubacterial sequences
aligned with the MultiAlin alignment tool. The proteins used to define
ProDom 1624 are as follows: ANCA_CLOTM, cellulosome anchoring protein
from C. thermocellum; SLP1_CLOTM and SLP2_CLOTM, SLPs from
C. thermocellum; GUN_BACS6, endoglucanase from
Bacillus sp. strain KSM-635; SLAP_ACEKI, S-layer
glycoprotein from A. kivui; SLPH_BACBR, SLP from
Bacillus brevis; SLPM_BACBR, middle cell wall protein from
B. brevis; APU_THETU, type II pullulanase from T. thermosulfurigenes; XYNX_CLOTM, exoglucanase from C. thermocellum; XYNA_THESA, endoxylanase A from
Thermoanaerobacterium saccharolyticum; SLAP_BACSH, SLP from
Bacillus sphaericus; OMPA_THEMA, outer membrane protein A
from Thermotoga maritima; SLAP_THETH, SLP from
Thermoanaerobacterium ethanolicus; SLAP_BACAN, SLP protein
from Bacillus anthracis; SLAP_BACLI, SLP from Bacillus
licheniformis. The Th-APu sequences were manually fitted to the
ProDom alignment by introducing uniform gaps where necessary. White
characters on a black background indicate areas where sequence identity
is equal to or greater than 50%, while gray shading indicates areas
where sequence similarity is equal to or greater than 50%. The groups
of similar residues were considered to be I, L, V, M; D, E; N, Q; S, T;
R, K; and F, Y, W. Dots indicate gaps in the sequences. SLD1_THYDRO and
SLD2_THYDRO are the two Th-Apu SLDs.
-amylase-pullulanase from Bacillus sp. (18).
Previous studies have indicated that these enzyme-associated SLDs
may be responsible for the anchoring of proteins to the cell surface,
possibly by interacting with peptidoglycan in certain bacteria or with
the S-layer itself via SLH-SLH interactions (22, 31, 36).
Expression of the catalytic domain in E. coli.
Having
failed in our attempts to obtain a plasmid containing an intact
apuA gene, we constructed a plasmid containing only the PUL
domain of Th-Apu (pAPU
1). Expression trials using pAPU1 led to
the production of a significant amount of recombinant protein in
E. coli cells which was present in a soluble form in the
cytoplasmic fraction after cell lysis. Crude purification of this
protein could be achieved by a simple heat treatment (80°C, 30 min)
which provoked the precipitation of most of the contaminating proteins. Examination of the recombinant protein by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis revealed the presence of two
isoforms (the two species have identical N-terminal sequences) which
exhibit molecular masses of approximately 90 and 85 kDa. Zymogram
analysis was then employed, the results of which showed that both
species possess thermostable pullulanolytic activity, indicating that the catalytic determinants are indeed present within the PUL domain.
Conclusion. On the basis of our results, we propose that Th-Apu forms part of a maltose transport operon different from the one previously described for T. litoralis (15). This enzyme is probably secreted in T. hydrothermalis cells, although rather than being released into the extracellular medium, Th-APu may be anchored to the cell membrane or another hydrophobic component of the cell envelope via its TM domain, with its catalytic domain exposed to the surrounding medium. By analogy to other Pro-Thr-rich domains, it can be assumed that the PET domain has a rather extended, perhaps flexible, conformation which would be susceptible to proteolytic digestion (30). Thus, intense O glycosylation of this domain would reduce the vulnerability of this structure. With regard to its function, the PET domain may serve as a linker and/or perform a role similar to that of the Thr-rich region of the glucoamylase from A. awamori or may fulfill a cell wall-anchoring role rather like the Gly/Ser-rich region in SbsB (36). The role of the Th-Apu SLDs is unclear since, to our knowledge, nothing is known concerning the cell envelope of T. hydrothermalis. However, by analogy to other proteins which exhibit SLH motifs, we speculate that these domains interact with a hitherto unidentified component of the cell envelope.
Clearly, the idea that Th-Apu is localized at the cell surface appears to contradict previous experimental data from chemostat cultures which have indicated that Th-Apu is completely secreted and released into the extracellular medium. However, as others have already suggested (20, 26), it is possible that cellular attachment may be a transient state prior to complete release of the protein and/or that the prevailing conditions of a chemostat culture may provoke the degradation of the cell envelope, thus eliminating certain protein attachment points (e.g., SLH anchoring determinants).Nucleotide sequence accession number. The GenBank accession number for the 5.2-kb DNA fragment described here is AF113969.
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ACKNOWLEDGMENTS |
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We thank Jean-Claude Pernollet and Jean-Claude Huet for their assistance with the N-terminal protein sequencing and Béatrice Hermant for her general technical assistance.
This research forms part of a scientific program which was funded by the Europol'Agro consortium.
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FOOTNOTES |
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* Corresponding author. Mailing address: Unité de Physicochimie et Biotechnologie des Polymères, Institut National de la Recherche Agronomique, Université de Reims Champagne-Ardenne, Bât. 18, Moulin de la Housse, BP 1039, 51687 Reims Cedex 02, France. Phone: (33) 3 26 91 32 24. Fax: (33) 3 26 91 38 87. E-mail: odonohue{at}lille.inra.fr.
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