Detailed characterization of the enzyme showed that PelB is highly thermo-active and thermostable, with a melting temperature of 105C and a temperature optimum of 80C, the highest descri
Trang 1exopolygalacturonase from the hyperthermophilic
bacterium Thermotoga maritima
Leon D Kluskens1, Gert-Jan W.M van Alebeek2, Jasper Walther1, Alphons G.J Voragen2,
Willem M de Vos1and John van der Oost1
1 Laboratory of Microbiology, Wageningen University, the Netherlands
2 Laboratory of Food Chemistry, Wageningen University, the Netherlands
Pectin is a complex polysaccharide present in the cell
wall of higher plants, where it forms a network by
embedding the other cell wall polysaccharides cellulose
and hemicellulose The backbone of the pectin
mole-cule mainly consists of (partly methylated)
homogalac-turonan, interspersed with rhamnogalacturonan units,
which often contain sugar side chains composed of
arabinan and galactan [1]
Degradation of the pectin polymer occurs via a set
of pectinolytic enzymes, which can roughly be divided
into esterases, which remove ferulic acid, methyl or acetyl groups, and depolymerases The latter can be classified into lyases (b-elimination) and hydrolases [2] All hydrolases involved in degradation of pectin are classified as members of family 28 of the glycoside hydrolases, including the endopolygalacturonases, exo-polygalacturonases and rhamnogalacturonases [3,4] Although a handful of endopolygalacturonases, gener-ally of fungal origin [5–10], and a single rhamnogalac-turonase [11] have been the object of crystallization
Keywords
exopolygalacturonase; hydrolytic; mode of
action; pectin; thermostable
Correspondence
J van der Oost, Laboratory of Microbiology,
Wageningen University, Hesselink van
Suchtelenweg 4, 6703 CT Wageningen,
the Netherlands
Fax: +31 317 483829
Tel: +31 317 483108
E-mail: john.vanderoost@wur.nl
(Received 28 July 2005, accepted 24 August
2005)
doi:10.1111/j.1742-4658.2005.04935.x
An intracellular pectinolytic enzyme, PelB (TM0437), from the hyperther-mophilic bacterium Thermotoga maritima was functionally produced in Escherichia coliand purified to homogeneity PelB belongs to family 28 of the glycoside hydrolases, consisting of pectin-hydrolysing enzymes As one
of the few bacterial exopolygalacturonases, it is able to remove monogalac-turonate units from the nonreducing end of polygalacmonogalac-turonate Detailed characterization of the enzyme showed that PelB is highly thermo-active and thermostable, with a melting temperature of 105C and a temperature optimum of 80C, the highest described to date for hydrolytic pectinases PelB showed increasing activity on oligosaccharides with an increasing degree of polymerization The highest activity was found on the pentamer (1000 UÆmg)1) In addition, the affinity increased in conjunction with the length of the oligoGalpA chain PelB displayed specificity for saturated oligoGalpA and was unable to degrade unsaturated or methyl-esterified oligoGalpA Analogous to the exopolygalacturonase from Aspergillus tubin-gensis, it showed low activity with xylogalacturonan Calculations on the subsite affinity revealed the presence of four subsites and a high affinity for GalpA at subsite +1, which is typical of exo-active enzymes The phy-siological role of PelB and the previously characterized exopectate lyase PelA is discussed
Abbreviations
PelB, exopolygalacturonase B; PelA, exopectate lyase A; PGA, polygalacturonic acid; (GalpA) n , oligogalacturonate with degree of
polymerization n; DP, degree of polymerization; HPSEC, high-performance size-exclusion chromatography; HPAEC, high-performance anion-exchange chromatography.
Trang 2experiments, a 3D structure of an
exopolygalacturo-nase is not yet available
Exo-acting polygalacturonases generally cleave the
homogalacturonan part of pectin from the
nonreduc-ing end Exopolygalacturonases (EC 3.2.1.67) are
pro-duced by fungi and plants and catalyse the hydrolytic
release of monogalacturonic acid The mostly bacterial
exo-poly a-galacturonosidases (EC 3.2.1.82) liberate
digalacturonic acid residues from galacturonan [2,3]
In recent years, many (hyper)thermophilic organisms
have been described with the main emphasis on their
capacities to degrade starch and cellulose [12,13]
Although amply present in nature, pectin-degrading
(hyper)thermophiles have received relatively little
attention [14–19] Considering their thermostability
and activity, as well as their slightly acidic pH
opti-mum, galacturonases from these organisms are
believed to have potential in processes for clarifying
fruit juices Up to now, only a few thermostable
pec-tinolytic enzymes have been characterized in detail
[20–22]
The hyperthermophilic bacterium Thermotoga
mari-tima is able to grow on a large variety of simple and
complex carbohydrates, such as glucose, maltose,
starch, laminarin, xylan and cellulose [23,24] In
addi-tion, we recently reported on its ability to use pectin
as a carbon source [20] The T maritima genome
sequence revealed the presence of at least two
pec-tinase-encoding genes [25] One of these, PelA, has
been characterized in detail as an extracellular
exopec-tate lyase, releasing unsaturated trigalacturonate as the
major product [20] We here report on the
overproduc-tion, purification and characterization of an
exopoly-galacturonase from T maritima, hereafter referred to
as PelB In addition, the physiological role and
expec-ted synergy between the two pectinolytic enzymes of
T maritimawill be discussed
Results
Molecular characterization of PelB
The pelB gene (locus number TM0437) was identified
in the T maritima genome and annotated as a putative
exo-poly a-d-galacturonosidase [25] pelB is 1341 bp in length, which corresponds to a protein with a mole-cular mass of 50 kDa The highest sequence similarity
at amino-acid level (69%) was found with an annota-ted glycoside hydrolase from Bacillus licheniformis, the genome sequence of which has been published recently [26] The absence of a clear signal sequence consensus indicates that the enzyme’s localization is most likely cytoplasmic [27] pelB is positioned in the same gene cluster as the previously described pelA gene [20] (Fig 1) Comparative gene analysis with the aim of examining the distribution of pelB homologs demon-strated no conservation in genome environment com-pared with other completely sequenced genomes The tight clustering with seven surrounding genes in the same transcriptional direction (TM0436-443), with no
or small intergenic regions, suggests that pelB may be transcribed polycistronically (Fig 1) PelB belongs
to the large family 28 of the glycoside hydrolases consisting of endopolygalacturonases (EC 3.2.1.15), exopolygalacturonases (EC 3.2.1.67), exo-poly a-galac-turonosidases (EC 3.2.1.82), and rhamnogalacturonases (EC 3.2.1.-) [4] All 3D structures known from family
28 glycoside hydrolases adopt a so-called parallel b-helical structure, in which the catalytic domain con-sists of three or four b-strands⁄ coil (7–12 in total), resulting in three or four parallel b-sheets By using clustalxa multiple sequence alignment was made for the right-handed parallel b-helix domain of a selection
of family 28 members (Fig 2) Independently, we modeled PelB on EPG2, an endo-active polygalacturo-nase from Erwinia carotovora with low amino-acid identity (23%), using the fold-recognition server of 3D-PSSM [28] The 3D structure of the b-helix of EPG2 has been elucidated [8] Sequence conservation predominantly occurs in the regions flanking both catalytic aspartate residues (Asp239 and Asp260, PelB numbering), as well as the residues Asp261 and His296, believed to be of importance in the catalytic process, and Arg327 and Lys329, which may play a role in substrate binding (Fig 2) [6]
The predicted secondary structure of PelB corres-ponds closely to that of E carotovora EPG2, with only
a few exceptions Like EPG2, the parallel b-helix
Fig 1 Schematic organization of the pectinase gene cluster in T maritima (TM0433-0443) pelA and pelB are shown as grey arrows Adja-cent genes are a-glucuronidase (agu), acetyl xylan esterase (axe), Zn 2+ -containing alcohol dehydrogenase (adh), 6-phosphogluconate dehy-drogenase, decarboxylating (gnd), transcriptional regulator (GntR), oxidoreductase (ord), gluconate kinase (glk), two conserved hypothetical proteins (hyp1 and 2) Intergenic spacing with putative promoter regions is indicated by (D).
Trang 3comprises 10 complete turns PelB contains a few
inserted b-strands (1a in Fig 2), and one large insert
of 15 amino acids is present before the first b-sheet of
coil 3, which is on the edge of the pronounced
sub-strate-binding cleft in EPG2 (Fig 2)
Expression and purification
The 1341-bp pelB gene was cloned into a pET24d
vector as an NcoI⁄ BamHI fragment, resulting in
pLUW741 Introduction into E coli BL21(DE3)
resulted in the overproduction of the 50-kDa PelB, which was verified by SDS⁄ PAGE analysis The enzyme was purified to homogeneity by heat treat-ment of the cell-free extract, followed by anion-exchange chromatography, during which the protein was eluted at 0.6 m of NaCl Analysis of PelB by gel filtration resulted in a peak with an estimated mass of 212 kDa, corresponding to results of SDS⁄ PAGE analyses of the unboiled sample, sug-gesting that the configuration of PelB is a tetramer (not shown)
PB3 1PB1 PB2 PB3 eee ee eeee eee hhh TmarPelB (exo,-1):(40) -TDCSESFKR AI EELSKQG G GR L V PE G - V FLTGP I HLKSN I ELH V KG TI KFIP D PER Y LPVVLTR -FEG -IELYN : 81
EEEE EE EEEE EEEE HHHH EEE Ecaropg (endo) :(45) -TATSTIQK AL NNCDQ G KA V L SA G STS V FLSGP L SLPSG V SLL I DKGV TL RAVN N AKS F ENAPSSC-GVVDK NGK- : 86
EchrpehX (exo,-2):(164) TLNTSAIQK AI DACPT G CR I V PA G - V FKTGA L WLKSD M TLN L LQGA TL LGSD N AAD Y PDAYKIY-SYVSQVRPASLLN : 203
RsolPehC (?) :(140) FDSRPAFTA AI AACNAAG G GR V V PA G N WYCAGP I VLLSH V HFH L GADC TI YFSP N PDD Y AKDGPVDCGTNGKLYYSRWQS : 182
Thther (?) :(192)-SSGTLNTAAIQK AI DKCPD G GV V V PA G K I FVTGP I HLKSN M TLD V EG TL LGTT D PDQ Y PNPYDTDPSQVGQ-KSAPLIS : 235
AtubpgaX (exo,-1):(59) -DDSDYILS AL NQCNH-G G KV V FDEDKEYI I GTALN M TFLKN I DLE V LG TI LFTN D TDY W QANSFKQ GFQN - : 101
Athaepg (?) :(79) DSKTDDSAAFAA A WKEACAA- G ST I V PK G EYM V ESLEFKGPCKGP -V TL ELNG N FKAPATV - : 124
2.1 1a 2 3 eeee e eee eee TmarPelB : -YSPL VYALDCE N VA I G G -V LDG SADNEHWW -PWKGKK-DFGWKEGLPNQQEDVKKLKEMA - : 170
EEEE EEE EEE H HHHHHH HH EE Ecaropg : GCDAFITAVSTT N SG I G G -T IDG QGGVKLQ -DKK VSWWE-LAA D AK-VKKLKQN - : 172
EchrpehX : A IDKNSS-AVGTFK N IR I G G -I IDG NGWKRSA -DAKDELGNTLPQYVKS D NSKVSK DGI - : 298
RsolPehC : NDCLNYGAPIYARNQS N IA L G G DSSV LNG QAMTPFAGSGNTSMCWWTFKGTKGAYGVVDASTPSQASG N PNNVDLRTAAPGIADALYAKLTDPATPW : 302 Thther : T VSTDVYGNTIQYQ N IR I G G -V ING NGWAQVSS -KDTSVPIDDQFDQYQKG N SSNISTTAKNH - : 333
AtubpgaX : -ATTFFQLGGE D VN M G G -T ING NGQVWYD -LYAED D LI - : 165
Athaepg : -KTTKPHAGWI D FENIADF -T LNG NKAIFDG -QGSLAWKAN D CAKTGKCNSLP - : 188
3.1 1a 2 3 4.1 1a 2 3 5.1 1a eeee e eeee eeee eeee e eeee eeee eeee e TmarPelB : -ERGTPVEERVFG -KGHYLR -PSF V QFYRCRNVL V EGVKIINS P W CVHPVLSENVIIR -N I EISSTGP N DG IDPESCK : 196 EEEE EEEE EEEE EEEE EEEE EEEE EEEE Ecaropg : -TP R -L I QINKSKNFT L YNVSLINS P F HVVFSDGDGFTAWK TT I KTPSTAR N DG IDPMSSK : 180 EchrpehX : -LAKNQVAAAVATGMDTKTAYSQ R RSSL V TLRGVQNAY I ADVTIRN- P ANHGIMFLESENVVENS V I HQTFNAN N DG VEFGNSQ : 330 RsolPehC : QQDQNYLPALSEAGVAVAQRIFG -KGHYL R -PCM V EFIGCTNVL M ETYRTHAT P W QHHPTDCTNVVIRG - V TVDSIGP N DG FDPDACD : 356 Thther : -LALNQFNKYSSQG TSNAYAT R -SNL M VFNNVNGLY I GDGLIVTN P F HTISVSNSQNVVLNQ L I ASTYDCN N DG IDFGNST : 362 AtubpgaX : -L R -PIL M GIIGLNGGT I GPLKLRYS P Y YHFVANSSNVLFDGIDISGYSKSDNEAK N DG WDTYRSN : 174 Athaepg : -IN I RFTGLTNSK I NSITSTNSKL F HMNILNCKNITLSDIG I DAPPESL N DG IHIGRSN : 195 * 2 3 6.1 1a 2 3 7.1 1a 2 3 8.1 eeee eee eeeee ee eeee eeee eee ee eeee eeee eeee TmarPelB : Y M I EKCRFD TGDD SVIKSGRDADGRRIGVPSEYILV RDNLVISQAS HG GLVIGSEMSGG V RN V VAR N -NVYMNVERA LR L KT NSR - : 310
EEEE EEE EEEEE EE EEEE EEE EEE EE EEEE EEEE EEEE Ecaropg : N I I AYSNIA TGDD N AIKAYKGR -AETRNIS I LHNDFG TG HG -MSIGSE-TMG V YN V TVD D -LKMNGTTNG LR I KS DKS - : 286
EchrpehX : N I V FNSVFD TGDD S NFAAGMGQDAQKQ-EPSQNAW L FNNFFR HG HG AVVLGSHTGAG I VD V LAE N -NVITQNDVG LR A KS APA - : 442
RsolPehC : N V LCEGMTFN TGDD C AIKSGKNLDTAYG PAQNHV I QDCIMN SG HG GITLGSEIGGG V QQ I YAR N LTMRNAFYATNPLNIA IR I KT NMN - : 466
Thther : G L V VNSVFN TGDD D NFDAGVGLSGEQN-PPTGNAW V FDNYFG RG HG VIAMGSHTAAW I QN I LAE D -NVINGTAIG LR G KS QSG - : 475
AtubpgaX : N I I QNSVINN GDD C SFK -PNSTNIL V QNLHCN GS HG -ISVGSLGQYKDEVDIVE N VYVYNIS MFNASDMA R K VWPGTPSALS : 280 Athaepg : G V L IGAKIK TGDD C SIGDG -TENLI V ENVECG PG HG -ISIGSLGRYPNEQPVKGVTVRK -CLIKNTDNG VR I KT WPG - : 297
** 1a 2 3 9.1 1a 2 3 10.1 eee eeee eeee eeee eee eeee eee eee TmarPelB : -RG G YMENIFFIDNVAV N VSE -EVIRINLR Y DNEEGEYLPVVR -SVFVK N LKATGGK -YAVRIEG L : 350 EEE EEEE EEEE EEEE EEE EEEE EEE EEEEE Ecaropg : -AA G VVNGVRYSNVVMK N VAK PIVIDTV Y EKKEGSNVPDWS -DITFK D VTSETKG VVVLNG - : 326
EchrpehX : -IG G GAHGIVFRNSAMK N LAK -QAVIVTLS Y ADNNGTIDYTPAKVPARFYDFTVK N VTVQDSTGSNPAIEITGDSS : 482 RsolPehC : -RG G YVRDFHVDNVTLP N G -VSLTGAG Y GSGLLAGSPINSSVPLGVGARTSA N PSASQGGLITFDCDYQP-AK : 513 Thther : -NG G GARNITFRDSALAYITDNDGSPFLLTDG Y SSALPTDTSNWAPDEPTFHDITVE N CTVNGSK KYAIMFQG A : 515 AtubpgaX : ADLQGGGGS G SVKNITYDTALID N VDWAIEIT QC Y GQKN-TTLCNEYPSSLTISDVHIK N FRGTTSGSEDPYVGTIVCSS : 338 Athaepg : -SPPGIASNILFEDITM D NVS -LPVLIDQE Y CPYGHCKAGVPS -QVKLS D VTIKGIKG TSATKVAV : 341 2 3 11.1 2 eeeeee ee eee eeee TmarPelB : ENDYVKDILISDT IIEGAKISVLLEFGQLGMENVIMN - : (16)
EEEEEE EE EEEEE EEE Ecaropg : ENAK-KPIEVTMK -NVKLTS-DSTWQIKNVNVKK - : (-)
EchrpehX : KDIWHSQFIFSNMKL SGVSPTSISDLSDSQFNNLTFS - : (26)
Thther : PDGFDYNITFNNVFFG-AGTYQTKIYYLKNSTFNNVVFYG - : (538)
AtubpgaX : PDTCSDIYTSNINVTSPDGTNDFVCDNVDESLLSVNCTATSD : (-)
Athaepg : KLMCSKGVPCTNIAL SDINLVHNGKEGPAVSACSNIKP - : (19)
Fig 2 Multiple sequence alignment of parallel b-helix segment of family 28 glycoside hydrolases Sequences (GenBank identifier): PelB
T maritima (AAD35522.1), EPG2 Erwinia carotovora (CAA35998.1), PehX Erwinia chrysanthemi (AAA24842.1), Ralstonia solanacearum K60 PehC (AAL24033.1), PG Thermoanaerobacterium thermosulfurigenes (AAB08040.1), Pgx Aspergillus tubingensis (CAA68128.1), Pgx2 Arabi-dopsis thaliana (AAF21195.1) The mode of action (endo or exo) and the amount of GalpA cleaved off, respectively, are annotated in paren-theses A question mark indicates unknown activity mode The secondary structure is depicted for E carotovora polygalcturonase (in capitals, using Expasy’s Swiss model, entry 1BHE) and T maritima (small characters, derived from model based upon E carotovora 1BHE in the program 3D-PSSM) [28], for which E (e) indicates strand and H (h) helix The parallel b-strands (PB1, 1a, 2 and 3) forming 11 coils are shown for E carotovora and T maritima sequences, with the coil number printed in bold Catalytic residues are indicated by stars, and resi-dues presumed to be involved in substrate–subsite interaction are highlighted with arrows Insertions in PelB in comparison with EPG2 are printed in italics.
Trang 4Enzyme characteristics
PelB was examined by incubation with polygalacturonic
acid (PGA) following standard assay conditions The
experiments showed an increase in the amount of
reducing sugars ends, indicating that PelB is active on
PGA, the nonmethylated homogalacturonan part of the
pectin molecule Hydrolysis of PGA, analysed by
high-performance size-exclusion chromatography (HPSEC),
showed the initial formation of only monogalacturonic
acid, with a simultaneous decrease in length of PGA
(not shown) Therefore, PelB can be regarded as an
exo-acting polygalacturonase Highest activity using
PGA as a substrate was measured at 80C (Fig 3A),
making it the most thermo-active polygalacturonase
reported to date Differential scanning calorimetry
showed that PelB has a melting temperature of
105C (not shown) The pH optimum of PelB was
determined to be 6.4, making it slightly more alkali
than previously described polygalacturonases A
signifi-cant fall in activity was observed when the pH was
increased to 7 (Fig 3B) Zymogram experiments were
carried out with PelB and concentrated T maritima
medium fraction (supernatant) and cell extract using
PGA as a substrate These revealed that PelB is located intracellularly, as shown by a clear activity zone of the cytoplasmic fraction (not shown) No activity on PGA was observed when the corresponding medium fraction was concentrated and similarly analysed
Mode of action of PelB
To examine its mode of action in more detail, hydro-lysis products of oligogalacturonic acids generated
by PelB were analysed by high-performance anion-exchange chromatography (HPAEC) The initial reaction product of all substrates tested was monogal-acturonic acid (not shown), indicating that PelB is an exopolygalacturonase The activity on 0.25% (w⁄ v) PGA was found to be 6.1 UÆmg)1 over the first 2 h
A range of D4,5 unsaturated oligoGalpA species, containing a double bond between C4 and C5 at the nonreducing end, was incubated with PelB and ana-lysed by HPAEC Unsaturated (GalpA)3)5species were not hydrolysed by the enzyme As the unsaturated bond on this range of substrates is located at the nonre-ducing end, it can be concluded that PelB is attacking from the nonreducing end Moreover, fully methylated (GalpA)4)6 molecules were not hydrolysed by PelB, indicating that the presence of methyl esters prevents the enzyme from hydrolysing oligoGalpA Kester et al [29] found that the exopolygalacturonase from Asper-gillus tubingensis not only acts on the homogalacturo-nan part, but is also active on xylogalacturohomogalacturo-nan, a highly methyl-esterified backbone in which galacturonic acid units are highly substituted with xylose at position O-3 This prompted us to test this substrate as well On analysis by HPAEC, the formation of a d-galacturo-nate peak could be observed directly after addition of PelB, which is the result of its established galacturonase activity Only when high concentrations of PelB were used on xylogalacturonan (25 lgÆmL)1 rather than 3.2 ngÆmL)1 when assayed on PGA) was a minor amount of xylogalacturonate units detected in addition
to d-galacturonate (not shown)
Enzyme kinetics PelB activity was initially demonstrated using PGA as substrate As it seems highly unlikely that the cyto-plasmic PelB uses the large polymer as its natural substrate, kinetic parameters (Km and Vmax) were determined with saturated (GalpA)n (n¼ 2–8) PelB (8–16 ng in a reaction volume of 1 mL) and substrate (up to 12 mm) were incubated at 80C for 10 and
15 min Table 1 shows the kinetic parameters for PelB
Fig 3 Dependence of PelB activity on temperature (A) and pH (B).
Temperature (d) and pH optimum (m) were measured on PGA
fol-lowing standard assay conditions (see Experimental procedures).
Trang 5on GalpA ranging from digalacturonate to
octagalac-turonate For all concentrations, a typical Michaelis–
Menten equation was observed With an increasing
degree of polymerization (DP), the substrate affinity
increased significantly, up to 0.06 mm for (GalpA)8
The activity of PelB (Vmax) increased reaching a
plat-eau around 1000 UÆmg)1 at (GalpA)4, where kcat
val-ues seem to be independent when DP exceeds n¼ 4
Catalytic efficiency, kcat⁄ Km, increased constantly with
increasing DP, with a value for (GalpA)8 almost
30-fold higher than for (GalpA)2(Table 1)
Subsite mapping
On the basis of the assumptions of Hiromi [30] that
the intrinsic rate of hydrolysis (kint) in the productive
complex is independent of the length of the substrate,
Kmand Vmax were used to calculate the subsite
affinit-ies (see equations in Experimental procedures) The
subsite affinity An+1 (kJÆmol)1) was calculated for an
enzyme–substrate complex from n¼ 2–5 The intrinsic
rate constant kint was determined by plotting
exp(An+1⁄ RT) against (1 ⁄ kcat)n, which also allowed us
to calculate the binding affinity for subsite)1 The kint
value was found to be 262 s)1 Affinity values are
shown in Fig 4 as a schematic representation of the
subsite binding cleft of PelB The highest binding
affinity was found for the penultimate subsite +1
(40.2 kJÆmol)1), after which the affinity decreased
considerably when moving towards the reducing end
of the substrate, away from the catalytic site Along
with its exocleaving activity, thereby liberating
mono-galacturonic acid, the catalytic site of PelB should be
located in between subsites )1 and +1 (Fig 4)
Com-parative modeling previously showed that the binding
cleft of polygalacturonases can maximally hold eight
GalpA residues, resulting in a subsite order from)5 to
+3 [5] As the substrate most likely binds to the
non-reducing end towards the N-terminus of the enzyme
[31], this implies that PelB probably contains four sub-sites, from)1 to +3
Discussion
The pectinolytic hydrolase PelB from the hyper-thermophilic bacterium T maritima was heterologously produced and purified to homogeneity Detailed characterization of this enzyme is described in this paper, which is a continuation of the recent report of
an exopectate lyase (PelA) from the same organism [20]
Despite its clear exocleaving characteristics, the highest similarity at amino-acid level was found with family 28 endopolygalacturonases (EC 3.2.1.15), although it should be noted that the number of avail-able endopolygalacturonase sequences exceeds that for exocleaving galacturonate hydrolases The apparent absence of a signal peptide and the detection of pec-tinolytic activity in the cell fraction and not in the medium fraction supported our belief that PelB is cytoplasmic, in contrast with the majority of polygal-acturonases examined
Optimal activity on homogalacturonic acid was observed at 80C, making it the most thermoactive hydrolase active on this polysaccharide found to date Because of their catalytic and stability properties,
Table 1 Kinetic parameters of PelB from T maritima on saturated
oligogalacturonates (GalpA) with length n ¼ 2–8.
KM (m M )
Vmax (UÆmg)1)
kcat (s)1)
kcat⁄ K M (m M )1Æs)1)
Pentagalacturonate 5 0.24 1112 934 3892
Heptagalacturonate 7 0.07 1024 860 12288
Octagalacturonate 8 0.06 1003 843 14042
Polygalacturonate 170 0.06 1170 936 15600
Fig 4 Schematic representation of the subsite map of exopolygal-acturonase PelB A tetragalacturonate (GalpA) 4 has been modeled
in the binding site Subsites are numbered from )1 to +3, with the nonreducing sugar end facing the N-terminus of the enzyme Bind-ing affinity values are illustrated by bar diagrams The catalytic clea-vage site is indicated by an arrow.
Trang 6thermostable pectinolytic enzymes may be of great use
in industrial processes Considering its slightly acidic
pH optimum of 6.4, PelB may be useful in the fruit
juice industry, where it could be included in
clarifica-tion or colour extracclarifica-tion steps, which are often carried
out at elevated temperatures
Although bacterial exo-acting polygalacturonases
commonly generate digalacturonate, PelB was shown
to liberate monogalacturonic acid as the first and only
product on PGA and oligoGalpA On the basis of its
mode of action, PelB should be classified as an
exo-polygalacturonase (EC 3.2.1.67) To date, no crystal
structure of an exopolygalacturonase is available As
PelB has high similarity at the primary structure level
with endopolygalacturonases, especially around the
catalytic regions [3], we assume that the substrate
binds to the nonreducing sugar end moving towards
the N-terminus of the enzyme, as has been suggested
for endopolygalacturonases by Page`s and coworkers
[31] Perhaps the large insertion before coil 3 contains
residues that may play a role in obstructing the
sub-strate–subsite )2 interaction, although this insertion
seems to be absent from the exo-active A tubingensis
polygalacturonase Cho and coworkers [5] described
the amino acid residues in Aspergillus aculeatus
poly-galacturonase involved in hydrogen-bonding
inter-actions between the substrate-binding-cleft residues
and octaGalpA, and aligned the equivalent residues of
E carotovora EPG2 Two residues believed to be
involved in substrate binding at subsite)2 in E
caro-tovora EPG2, namely Arg152 binding the carboxy
group and Lys229 interacting with 2-OH, are also
con-served in PelB and A tubingensis
exopolygalacturo-nase Direct obstruction of a possible GalpA
interaction with its equivalent subsite)2 may therefore
be brought about by adjacent residues Although
phy-logenetically classified amongst the bacterial
endopoly-galacturonases [3], PelB displays characteristics that
clearly bear more resemblance to the group of fungal
exopolygalacturonases Obviously, the primary
struc-ture alone restricts us to explain PelB’s mode of
action in more detail Considering the homology
bet-ween exogalacturonases and endogalacturonases, the
difference in mode of action probably depends on
subtle changes in the catalytic and⁄ or
substrate-bind-ing region Unfortunately, only a few
exopolygalactu-ronases have been fully characterized and identified
and therefore the amount of available sequences is
limited
Exopolygalacturonases that liberate
monogalacturo-nate are generally produced by fungi and plants, with
the exception of one originating from the bovine
rumi-nal bacterium Butyrivibrio fibrisolvens [32] Like PelB,
this enzyme is localized intracellularly B fibrisolvens also contains an exopectate lyase that generates unsat-urated trigalacturonates, similar to PelA To our know-ledge, T maritima and B fibrisolvens are the only two bacteria described that contain such a similar combina-tion of pectinolytic enzymes, although the exopolygal-acturonase from B fibrisolvens was shown to degrade both saturated and D4,5 unsaturated oligoGalpA [33] Kinetic analyses have shown that PelB hydrolyses oligoGalpA very rapidly with an increasing affinity for longer oligoGalpA molecules The specific activity [reaching a plateau for (GalpA)4 at 1000 UÆmg)1] is among the highest known for polygalacturonases, and the highest of all oligoGalpA-active exohydrolases The highest affinity was found for the subsite +1 This high value is typical for exo-active hydrolytic enzymes, such as the exopolygalacturonase from A tubingensis and a barley b-d-glucosidase [34,35] The absolute value, however, (+40.2 kJÆmol)1) is much higher than has been reported previously for this subsite The rea-son for this may be the thermo-active character of the enzyme, which obliges PelB to bind its substrate tightly enough at high temperatures An affinity value closer
to mesophilic values may lead to a spontaneous disso-ciation of the substrate–subsite complex The intrinsic rate constant, kint, is rather low compared with the highest values found for kcat
Cho and coworkers tested kinetic models of octaga-lacturonate, using three polygalacturonases (including
A aculeatuspolygalacturonase), and concluded that the binding clefts in polygalacturonases can accommodate maximally eight GalpA residues at subsites from)5 to +3 [5] Along with the suggestions of Page`s and coworkers [31] that the GalpA binds to the nonreducing end moving towards the N-terminus of the enzyme, PelB can accommodate only four subsites in total, namely from )1 to +3, which was shown by the activity that reached a maximum at (GalpA)4 (Table 1) However, the catalytic efficiency factor (kcat⁄ Km) still increases with an increase in DP of the substrate, which would imply an extended substrate-binding region According
to this model, oligoGalpA exceeding a DP of 4 would comprise GalpA oligomers at the reducing end which are presumably exposed to the solvent region This pre-ference for longer oligoGalpA molecules seems to be in conflict with its cytoplasmic character and may perhaps
be due to conformational changes in the substrate, thereby facilitating binding to the substrate-binding cleft It is obvious that elucidation of the 3D structure of PelB would give more insight into structural organiza-tion of the binding site
T maritimacontains at least two evident pectinolytic enzymes PelA appears to be the only extracellular
Trang 7enzyme in T maritima able to depolymerize the
homo-galacturonic acid part of pectin into, predominantly,
unsaturated trigalacturonates [20] However, PelB’s
inability to degrade these intermediates suggests an
intermediate conversion of unsaturated oligoGalpA
Although the unsaturated oligoGalpA tolerated
high-temperature conditions without being degraded, other
in vivofactors besides temperature and pH may play a,
to date unclear, role in its stability Alternatively,
un-saturated oligoGalpA may be un-saturated by another, as
yet unidentified, pectinolytic enzyme To address
ques-tions such as these, we are currently using DNA
micro-array analyses to obtain insight into the complete set of
genes involved in pectin catabolism by T maritima
Experimental procedures
Organisms, growth conditions and plasmids
T maritimastrain MSB8 (DSM 3109) was grown at 80C
and pH 6.5 as described previously [20] The bacterial strain
used for the initial cloning experiments was E coli TG1
[supE hsd D5 thi D(lac-proAB) F¢ (traD35 proAB+
lacIq lacZ DM15)] E coli BL21(DE3) (hsdS gal (kclts 857 ind1
Sam7 nin5 lacUV5-T7 gene 1)) was used for heterologous
expression The plasmid used for recombinant work was
pET24d from Novagen (Madison, WI, USA)
PGA was obtained from ICN (Zoetermeer, the
Nether-lands) Saturated oligoGalpA (DP 2–8) and unsaturated
oligoGalpA (DP 3–7) were prepared and purified from
polygalacturonase and pectin lyase digestions as described
by van Alebeek et al [36] Methyl esterification of saturated
oligoGalpA [(6-O-CH3-GalpA)4)6] was carried out with
anhydrous acidic methanol [37] Modified hairy regions
were isolated from apple, saponified, and used as a source
of xylogalacturonan [38]
Recombinant DNA techniques
Genomic DNA of T maritima was isolated by using an
established protocol [39] Small-scale plasmid DNA
isola-tion was carried out using the Qiagen purificaisola-tion kit
(Valencia, CA, USA) DNA was digested with restriction
endonucleases and ligated with T4 DNA ligase, according
to the manufacturer’s specifications (Life Technologies,
Rockville, MD, USA) DNA fragments were purified from
agarose by QiaexII or from a PCR mix by using the PCR
purification kit (Qiagen) Chemical transformation of
E coli TG1 and BL21(DE3) was carried out using
estab-lished procedures [40]
The gene encoding an exopolygalacturonase (TM0437)
was identified in the course of the analysis of the T
mari-tima genome [25] Primers for gene amplification were
TGGAAGAAC (NcoI site in bold), and BG889 (antisense), 5¢-GCGTCACCTCGGATCCTTATTTCAGC (BamHI site
in bold) A PCR was carried out on 100 ng genomic DNA
of T maritima, following the procedure described previ-ously [20] After digestion with NcoI and BamHI, the gene product was cloned in a pET24d expression vector (Nov-agen) The resulting plasmid, pLUW741, was introduced into E coli TG1 and BL21(DE3)
DNA and amino-acid sequence analysis
Cloned PCR products were sequenced by the dideoxynucle-otide chain termination method [41] with a Li-Cor automa-tic sequencing system (model 4000L; Westburg, Leusden) DNA and protein sequencing data were analysed with the dnastar package and compared with the GenBank Data Base by blast [42] clustalx and genedoc were used for multiple alignment and subsequent adjustment of the exopolygalacturonase amino-acid sequence, respectively
Purification of PelB
E.coli BL21(DE3) harboring pLUW741 was grown over-night (37C, 150 r.p.m.) in a 5-mL TYK [1% (w ⁄ v) tryp-tone, 0.5% (w⁄ v) yeast extract, 0.5% (w ⁄ v) NaCl,
50 lgÆmL)1 kanamycin] preculture One milliliter was used
to inoculate 1 L TYK in a baffled 2 L Erlenmeyer flask After overnight growth at 37C at 120 r.p.m., the culture was centrifuged for 15 min at 8500 g at 4C, medium was discarded, and the cells were resuspended in 10 mL 20 mm Tris⁄ HCl, pH 8.0 The cell suspension was sonicated (3· 15 s), and cell debris was removed by centrifugation at
16 000 g for 10 min The resulting supernatant was incuba-ted for 20 min at 80C, and precipitated proteins were removed by an additional centrifugation step (16 000 g,
10 min) The heat-stable cell-free extract was loaded on to
an ion-exchange chromatography column (Q Sepharose; Amersham Pharmacia Biotech, Inc., Piscataway, NJ, USA), which was equilibrated with 20 mm Tris⁄ HCl, pH 8.0 Bound proteins were eluted by a linear gradient from
0 to 1 m NaCl in the same buffer Fractions containing PelB were pooled and concentrated (Filtron Technology Corp.; 30-kDa cut-off) Protein concentrations were spectrophotometrically calculated using the absorption coefficient Its native configuration was determined by run-ning PelB over a gel-filtration column (Superdex 200; Amersham Pharmacia Biotech, Inc.) and comparing it with
a set of marker proteins, using 20 mm Tris⁄ HCl ⁄ 100 mm NaCl, pH 8.0, as elution buffer
Enzyme assays and kinetics
PelB activity was measured by determining the formation
of reducing sugar end groups, using the Nelson–Somogyi
Trang 8assay [43] Standard assays were carried out at 80C in
1 mL 100 mm sodium phosphate buffer, pH 6.5, containing
0.25% (w⁄ v) PGA The reaction was started by the
addi-tion of an appropriate amount of PelB, and samples were
taken at regular time intervals The reaction was stopped
by adding 200 lL of the sample to a Somogyi reagent mix
and treated according to the protocol [43] Finally, the
sam-ple was analysed at 520 nm One enzyme unit (U) was
defined as 1 lmol reducing end groups released per minute
A 100 mm McIlvaine buffer was used for determining the
pH optimum of PelB
Kinetic constants were measured in duplicate under
opti-mal enzyme conditions (80C, pH 6.5) in a 30 mm
phos-phate buffer, using saturated oligogalacturonic acids with
a degree of polymerization (DP) of 2–8 [(GalpA)2 to
(GalpA)8] Substrate concentrations up to 12 mm
oligogal-acturonic acid were used, exceeding up to 10 times the Km
value Care was taken to measure initial reaction rates, and
the initial enzyme concentration was kept well below the
initial substrate concentration Km and Vmax were
calcula-ted using the Michaelis–Menten fit in table curve (SPSS
Inc., AISN Software) The turnover rate (kcat) was
calcula-ted from Vmax, using a calculated molecular mass of
50 483 Da for PelB The substrate specificity was examined
by measuring PelB activity on 1 mm saturated oligoGalpA
Enzyme reactions used for HPLC analyses were carried
out at 80C in 30 mm sodium phosphate buffer (pH 6.4)
PGA and xylogalacturonan (modified hairy regions) were
used at concentrations of 0.25% (w⁄ v), and (un)saturated
oligoGalpA and methylated oligoGalpA were used at an
end concentration of 2 or 2.5 mm PelB (4.6 ngÆmL)1) was
used in an incubation volume of 400 lL Samples (50 or
100 lL) were taken at time intervals, and reactions were
stopped by cooling on ice and by addition of 0.4 sample
volume of 50 mm NaOH, thereby increasing the pH to
8.0–8.5 Samples were stored at )20 C until analysed by
HPAEC
HPAEC analysis
HPAEC analysis at pH 12 was performed as described
pre-viously [37] Saturated and unsaturated oligoGalpA were
detected using a pulsed amperometric detector
(Electro-chemical Detector ED40; Dionex, Sunnyvale, CA, USA)
Pure saturated oligoGalpA species (DP 1–7) were used as
standards for external calibration of the system Product
formation was quantified by peak integration (Chromquest,
Thermoseparation Products, San Jose, CA, USA) Specific
activity [nmol productÆmin)1Æ(mg protein))1] was calculated
from the formation of saturated oligoGalpA over time
HPSEC analysis
HPSEC analyses were performed on three TSKgel columns
(7.8 mm internal diameter· 30 cm per column) in series
(G4000 PWXL, G3000 PWXL, G2500 PWXL; Tosohaas)
in combination with a PWX-guard column (Tosohaas, Stuttgart, Germany) Elution was carried out at 30C with 0.2 m sodium nitrate at 0.8 mLÆmin)1 The eluate was moni-tored using a refractive index detector Calibration was performed using dextrans, pectins and oligoGalpA
Differential scanning calorimetry
Thermal unfolding experiments were carried out on a Mic-roCal VP-DSC in the temperature range 50–125C at a heating rate of 0.5CÆmin)1 Enzyme samples were dialyzed against 50 mm sodium phosphate buffer, pH 6.5, before analysis
Calculation of subsite affinities
Subsite affinity values were calculated using the obtained kinetic data as described by Hiromi and coworkers [30,44] The subsite affinity An was calculated according to the equation:
lnðkcat=KmÞnþ1 lnðkcat=KmÞn¼ Anþ1=RT The parameter kcatwas derived from the maximum velocity (V), divided by the molar concentration of the enzyme (e0, included in Vmax) R and T are the gas constant and the temperature (in Kelvin), respectively The values A-1 and kintwere derived from a plot of exp(An+1⁄ RT) against (1⁄ kcat)n:
expðAnþ1=RTÞ ¼ ½kint=ðkcatÞn 1 expðA1=RTÞ
in which A-1 and kint are determined from the vertical and horizontal intercepts, respectively
Acknowledgements
We are very grateful to Dr J.A.E Benen and Ing H.C.M Kester for helpful discussions, and Ans Geer-ling for technical assistance
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