Galactomannan hydrolysis andmannosemetabolism inCellvibriomixtusMaria S.J. Centeno1, Catarina I.P.D. Guerreiro1, Fernando M.V. Dias1, Carl Morland2, Louise E. Tailford2,Arun Goyal1, Jose A.M. Prates1, Luıs M.A. Ferreira1, Rui M.H. Caldeira1, Emmanuel F. Mongodin3,Karen E. Nelson3, Harry J. Gilbert2 & Carlos M.G.A. Fontes1

1CIISA – Faculdade de Medicina Veterinaria, Polo Universitario do Alto da Ajuda, Lisboa, Portugal; 2Institute for Cell and Molecular Biosciences,

University of Newcastle upon Tyne, The Medical School, Newcastle upon Tyne, UK; and 3The Institute for Genomic Research, Rockville, MD, USA

Correspondence: Carlos M.G.A Fontes,

CIISA – Faculdade de Medicina Veterinaria,

Polo Universitario do Alto da Ajuda, Avenida

da Universidade Tecnica, 1300-477 Lisboa,

Portugal. Tel.: 1351 213652876; fax: 1351

213652889; e-mail: cafontes@fmv.utl.pt

Present address: Arun Goyal, Department of

Biotechnology, Indian Institute of Technology

Guwahati, North Guwahati, Guwahati 781

039, Assam, India.

Received 29 March 2006; revised 1 June 2006;

accepted 1 June 2006.

First published online June 2006.

doi:10.1111/j.1574-6968.2006.00342.x

Editor: Marco Moracci

Keywords

a-galactosidase; epimerase; galactomannan;

glycoside hydrolase; Cellvibrio .

Abstract

Galactomannan hydrolysis results from the concerted action of microbial endo-

mannanases, manosidases and a-galactosidases and is a mechanism of intrinsic

biological importance. Here we report the identification of a gene cluster in the

aerobic soil bacterium Cellvibrio mixtus encoding enzymes involved in the

degradation of this polymeric substrate. The family 27 a-galactosidase, termed

CmAga27A, preferentially hydrolyse galactose containing polysaccharides. In

addition, we have characterized an enzyme with epimerase activity, which might

be responsible for the conversion of mannose into glucose. The role of the

identified enzymes in the hydrolysis of galactomannan by aerobic bacteria is

discussed.

Introduction

The plant cell wall represents the most abundant reservoir of

renewable energy and carbon within the biosphere (Gilbert

et al., 2002). Mannan consists of a backbone of b-1,4-linked

mannose residues while in glucomannans the backbone

comprises both glucose and mannose residues randomly

distributed (Brett & Waldren, 1996). Galactose monomers

may decorate the mannose residues of both hemicelluloses

through a-1,6 linkages and therefore these polysaccharides are

usually referred to as galactomannans and galactoglucoman-

nans, respectively. Galactomannans are also storage carbohy-

drates in some plant seeds such as carob (Brett & Waldren,

1996). The hydrolysis of mannose containing polysaccharides

into its monomeric components requires the action of endo-

b-1,4-mannanases, exo-b-1,4-mannosidases and b-1,4-gluco-

sidases (McCleary, 1988; Puls & Schuseil, 1996).

In the sequence-based classification of glycoside hydro-

lases (GH) a-galactosidases are located in families 4, 27 and

36 (Coutinho & Henrissat, 1999). Family 4 is exclusively

prokaryotic, family 36 contains both bacterial and eukaryo-

tic enzymes, while family 27 comprises predominantly

eukaryotic GHs. While most eukaryotic a-galactosidases

are in family 27, the majority of prokaryotic enzymes have

been classified into families 4 or 36. Ultimately, the substrate

specificity of these enzymes is not restricted to the de-

branching activity of hemicelluloses, as a-galactosidases

have been shown to be crucial in other biological systems.

For example, removal of galactose from short chain galacto-

oligosaccharides, such as melibiose, raffinose and stachyose

that are carbohydrate reserves in leguminous plants, is

catalysed by plant and microbial a-galactosidases usually

located in families 4 and 36 (Fridjonsson et al., 1999). In

eukaryotes, lysosomal a-galactosidases are involved in the

catabolism of large macromolecules, such as glycoproteins

and glycolipids, and these enzymes belong to family 27

(Garman & Garboczi, 2004). Interestingly, most a-galacto-

sidases acting on branched hemicelluloses were shown to

FEMS Microbiol Lett 261 (2006) 123–132 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

have broad substrate specificities, also hydrolysing low

molecular weight substrates (Dey et al., 1993). There is,

however, a paucity of information relating to the molecular

determinants of substrate specificity in enzymes acting on

the removal of side chains from hetero-polymeric mannans.

The metabolism of the hydrolysis products of galactose

containing mannans and glucomannans, by aerobic soil

bacteria such as Cellvibrio mixtus, is poorly understood.

This is surprising, considering the detailed information that

is available on the hydrolysis of plant cell wall polysacchar-

ides. It is generally established that mannose residues are

phosphorylated by a transmembrane permease to mannose

6-phosphate and then converted to fructose 6-phosphate by

an intracellular phosphomannose isomerase (Binet et al.,

1998). In addition, galactose might be transformed into

galactose 1-phosphate by an intracellular galactokinase and

then converted into glucose 1-phosphate through the Leloir

pathway (Holden et al., 2003). These possibilities remain,

however, to be confirmed in Cellvibrio.

Here we report the cloning and the biochemical charac-

terization of two enzymes involved in the degradation and

metabolism of galactomannan, from the prolific plant cell

wall degrader Cellvibrio mixtus. It is shown that the family

27 a-galactosidase of Cellvibrio mixtus is a polymer specific

enzyme with very limited activity on galacto-oligosacchar-

ides of the raffinose series. In addition, CmEpiA is respon-

sible for the interconversion of mannose into glucose by

Cellvibrio mixtus.

Materials and methods

Bacterial strains, plasmids and cultureconditions

Escherichia coli strains used in this study were BL21(DE3),

Origami (Novagen) and XL1-Blue (Stratagene). The bacter-

iophages and plasmids used in this work were lZAPII

(Stratagene), pBluescript SK� (Stratagene), pGEM-T (Pro-

mega) and pET21a (Novagen) and pET28a (Novagen).

Cellvibrio mixtus (NCIMB 8633) was cultured aerobically at

20 1C in Dubos mineral salts medium or on Dubos agar

plates overlaid with filter paper (Millward-Sadler et al.,

1995). Culture conditions were maintained as described

previously (Millward-Sadler et al., 1995). The recombinant

proteins, encoded in pET21a and pET28a derivatives, were

expressed in E. coli BL21 and Origami strains, as described

below.

General recombinant DNA procedures

Transformation of E. coli, digestion of DNA with restriction

endonucleases, ligation of DNA with T4 DNA ligase, agarose

gel electrophoresis, Southern hybridization, slot blot and

plaque hybridizations were carried out as described pre-

viously (Berns & Thomas, 1965; Sambrook et al., 1989). The

genomic library was constructed in lZAPII using the

approach described by Clarke et al. (1991). The library was

plated on NZYM top agar at a density of three plaques per

cm2 and screened for a-galactosidase activity using the

methylumbelliferyl a-D-galactosidase overlay technique.

DNA hybridizations were performed using the fluorescein

system from Amersham according to the manufacturer’s

protocol. Two inverse PCR amplifications were performed

to clone the full-length sequence of the genes located

upstream from aga27 (encodes CmAga27A). Briefly, for the

first inverse PCR experiment, Cellvibrio mixtus genomic

DNA was digested with the enzyme EcoRI and resulting

DNA fragments ranging from 4.5 to 5.5 kb were eluted from

agarose gels and religated. In the second experiment, the

enzyme used was PstI and the fragments ranging from 1.5

and 2.5 kb were ligated. The primer pairs used in the first

and second amplifications were as follows: first amplifica-

tion (EcoRI), 50-GCAGGTCCATAAACATACGC-30 and 50-

GGTTGGCCAATATTTTGCGC-30; second amplification

(PstI), 50-CAATGACAGATGAAGGAAGC-30 and 50-CTTAG

CGCTATTGCTGATTG-30. PCRs were performed using 1 U

of the thermostable DNA polymerase pFU turbo (Strata-

gene) and following the manufacturer’s instructions. PCR

products were cloned into pGEM-T (Promega) and the

resulting plasmids were named pZC3 and pZC4, respec-

tively. The nucleotide sequence of DNA was determined

with an ABI Prism Ready Reaction DyeDeoxy terminator

cycle sequencing kit and an Applied Biosystems 377A

sequencing system.

Expression and purification of recombinantproteins

To express CmAga27A, CmEpiA and CmUnkA in E. coli, the

DNA encoding the mature enzymes were amplified by PCR

from Cellvibrio mixtus genomic DNA using the thermo-

stable DNA polymerase pFU Turbo (Stratagene). The pri-

mers used for the amplifications incorporated designed

restriction sites (Table 1). The PCR products were cloned

into pGEM-T and sequenced to ensure that no mutations

had occurred during PCR. The recombinant derivatives of

pGEM-T containing the Cellvibrio mixtus genes were

digested with the required restriction enzymes (Table 1)

and the excised DNA fragments were cloned into the

similarly digested vector. The three recombinant proteins

contain N- or C-terminal His6-tag. The resulting plasmids

encoding recombinant CmAga27A, CmEpiA and CmUnkA,

were named pZC5, pZC6 and pZC7, respectively.

Escherichia coli BL21 harbouring pET derivatives encod-

ing CmEpiA and CmUnkA was cultured in LB contain-

ing 100 mg mL�1 ampicillin at 37 1C to mid-exponential

phase (A550 nm 0.6) at which point isopropyl-b-D-thiogalacto-

FEMS Microbiol Lett 261 (2006) 123–132c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

124 M.S.J. Centeno et al.

pyranoside (IPTG) was added to a final concentration of

1 mM and the cultures were incubated for a further 5 h.

Recombinant CmAga27A formed inclusion bodies when ex-

pressed under those conditions using E. coli BL21. Therefore,

the pET21a derivative, pZC5, encoding the Cellvibrio mixtus

a-galactosidase was transformed into E. coli Origami. Recom-

binant E. coli was cultured as described above but after

induction the cells were maintained at 16 1C for 16 h. Cells

were pelleted by these cultures by centrifugation and the

bacterial pellets were resuspended in 50 mM sodium HEPES

buffer, pH 7.5, containing 1 M NaCl and 10 mM imidazole.

The buffer of CmAga27 also contained 5 mM dithiothreitol.

The three recombinant proteins were purified by immobilized

nickel affinity chromatography as described previously (Car-

valho et al., 2004) and exchanged into the buffers described

below.

Enzyme assays

The activity of CmAga27A was routinely determined with

10 mM p-nitrophenyl a-D-galactopyranoside (pNPaGAL),

at 37 1C, in 25 mM Tris-HCl buffer, pH 8.5 (pH optimum of

the enzyme), containing 5 mM dithiothreitol. To screen for

activity against 4-nitrophenyl-glycosides, substrate and en-

zyme concentrations were 10 mM and 1 mM, respectively,

and assays were carried out for up to 1 h. One unit of

enzyme for pNPaGAL was defined as the amount of enzyme

that liberates 1 mmol of p-nitrophenol per min from pNPa-

GAL. The pH profile of the a-galactosidase was determined

with 10 mM pNPaGAL using the following buffers: pH

6–8.5, 50 mM phosphate; pH 8.5–9.5, 50 mM Tris-HCl.

The resistance of the enzyme to proteinase and thermo-

stability were determined as described previously (Fontes

et al., 1995). Activity of CmAga27A against oligosaccharides

of the raffinose series and polysaccharides was determined

by incubating 5 mg of enzyme, in 25 mM Tris-HCl buffer, pH

8.5, containing 5 mM dithiothreitol, with the appropriate

substrate [1% (w/v)] in a volume of 100mL and released

reducing sugar was measured with the Somogyi–Nelson

assay (Nelson, 1944; Somogyi, 1952). To determine the

kinetic parameters of the enzyme against its target sub-

strates, an appropriate dilution of the enzyme was incubated

with concentrations of substrate ranging from 0.05 to

10 g L�1 for polysaccharides and 0.01–10 mM for pNPaGAL.

Activities were measured in the linear phase of the enzyme

reactions. All reported results are the mean of three

replicates.

Phosphomannose isomearse activity (converts mannose

6-phosphate to fructose 6-phosphate) was measured using a

coupled enzymatic assay utilizing phosphoglucose isomer-

ase (converts fructose 6-phosphate to glucose 6-phosphate)

and glucose 6-phosphate dehydrogenase (oxidizes glucose

6-phosphate and reduces NADP 1 to NADPH). Mannose

6-phosphate was used as the substrate and the progress of

the reaction was followed spectrophotometrically at 340 nm

by measuring the production of NADPH (Todd & Tague,

2001). To confirm the validity of the assay, the increase in

absorbance at 340 nm was monitored in a reaction contain-

ing 0.6 units of a commercial phosphomannose isomerase

(Sigma p-5153). The reaction mixture consisted of 27 mM

Tris-HCl, pH 7.5, containing 5 mM MgCl2, 1 mM b-NADP,

1 U mL�1 glucose 6-phosphate dehydrogenase (Sigma G-

8289) and 0.5 U mL�1 of phosphoglucose isomerase (Sigma

P-5381). The reaction was started by adding 3 mM of

mannose 6-phosphate (Sigma M-6876) and was monitored

at 37 1C in an Ultrospec III spectophotometer (Pharmacia)

using the enzyme kinetic module of Biochrom Ltd software

package v. 1.01 (Pharmacia). Mannokinase activity was

measured using, essentially, the assay system described

above but using mannose as the substrate and Sigma

phosphomannose isomerase to convert mannose 6-phos-

phate into fructose 6-phosphate. To measure the kinetic

parameters of CmEpiA, 25 mg of enzyme were incubated

with 5–500 mM mannose in a volume of 100mL at 37 1C in

the buffer conditions described above. Glucose production

was monitored at various time points (up to 30 min) with

the D-Glucose-HK kit from Megazyme, using a final ATP

concentration of 20 mM. Optimum pH and temperatures

were determined as described above.

Results

Cloning and sequencing of Cellvibrio genes

A Cellvibrio mixtus genomic library, prepared in lZAPIII,

was screened for a-galactosidase activity. Four a-galactosi-

dase positive clones were isolated from the library and

Table 1. Primers, vectors and restriction sites used to hyperexpress CmAga27A, CmEpiA and CmUnkA in Escherichia coli

Gene Restriction sites Vector Primers

aga27A NdeI pET21a 50CTCCATATGCAAAAATTTGAGCATCTC

XhoI 50CACCTCGAGCTGCGGTGTTAAACGCAA

epiA NheI pET28a 50CTCGCTAGCAACGCCGCCATGCTTG

XhoI 50CACCTCGAGTTAAGTTGTGTGTAC

unkA NheI pET21a 50CTCGCTAGCTTTAAAGAAAAAGCAAAA

XhoI 50CACCTCGAGAACCAAATCCTTCATTAC

Bold type represents restriction sites designed in primers.

FEMS Microbiol Lett 261 (2006) 123–132 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

125Galactomannan utilization by Cellvibrio mixtus

purified to homogeneity by replating on the host strain. The

corresponding recombinant plasmids were excised from the

isolated clones and rescued into E. coli cells which displayed

activity against pNPaGAL (not shown). Restriction map-

ping and DNA hybridization studies revealed that all iso-

lated DNA fragments comprise the same 4.7-kb Cellvibrio

mixtus genomic region (not shown) and this plasmid was

named pZC2. The isolated DNA fragment was sequenced

revealing the presence of an ORF of 1215 bp, which was

designated aga27A. The gene encodes a polypeptide of 405

residues, named CmAga27A, with a predicted Mr of 45819.

The region upstream aga27, which revealed the presence of

an incomplete ORF, was cloned using the inverse PCR

approach described previously (Fig. 1a). The derived se-

quence comprised a cluster of three genes of 1179, 1230 and

1368 bp, which were designated unkA, epiA and man5A,

respectively (Fig. 1b). The intercistronic regions separating

unk-epiA and epiA-man5A were of 3 and 12 bp, respectively.

The proteins encoded by these three genes were designated

CmUnkA, CmEpiA and CmMan5A, respectively, and dis-

played predicted Mrs of 44188, 46524 and 51620. The

nucleotide sequence of the 7-kb sequence characterized in

this report appears in GenBank with the accession number

AY526725. Southern hybridization using aga27A as the

probe showed that a single copy of the gene is present in

the bacterial genome (data not shown). Two putative

promoters were identified upstream of unkA, epiA, man5A

and aga27A, with � 10 and � 35 well conserved regions

(Fig. 1c). In addition, two regions of dyad symmetry were

identified 18 and 22 nucleotides downstream of the putative

translational stop codon of man5A and aga27A, respectively

(Fig. 1b). The two 26-bp regions have the potential to form a

substantial stem–loop structure, characteristic of a r-inde-

pendent transcription terminator sequences.

Comparison of the primary sequence of CmAga27A with

biological databanks, searched by BLAST (www.ncbi.nlm.

nih.gov/BLAST), revealed that the protein display consider-

able sequence similarity with GH27 enzymes. Sequence

alignments (Fig. 2a), show that CmAga27A displays the

highest identities (4 40%) with GH27 putative enzymes

from Microbulbifer degradans (ZP_066516), Saccharopoly-

spora erythraea (AAC99325), Streptomyces avermitilis

(NP_822650) and a-galactosidases CjAga27A and Aga27A

from Cellvibrio japonicus and Clostridium josui, respectively

(BAB83765). Interestingly, GH27 enzymes presenting lower

identity scores with CmAga27A (below 47%) are all from

eukaryotic origin. It has been shown that in GH27 substrate

specificity is mediated by the nature of the b5–a5 loop,

which was recently called the substrate recognition loop

(Fujimoto et al., 2003). In the six GH27 bacterial enzymes

the presence of cysteine and tryptophan residues in the loop,

which coordinate the 2-hydroxyl of galactose in plant and

fungi a-galactosidases, suggest that all the bacterial enzymes

are a-galactose specific (Fig. 2a). A phylogeny tree of the 36

GH27 enzymes with demonstrated a-galactosidase activity

(see CAZy database), reveals that prokaryotic and eukaryotic

proteins have evolved from a common precursor, although

mammalian, plant, fungi and bacterial sequences segregate

into distinct clusters (Fig. 2b). In common with most a-

galactosidases, CmAga27A displays no evidence of modular

architecture or delineating linker sequences. In addition,

CmAga27A contains a typical signal peptide in which two

N-terminal basic residues are followed by 20 small hydro-

phobic amino acids capable of forming a a-helical structure,

with cleavage predicted to occur between Ala-23 and Gln-24.

In a previous report, it was demonstrated that CmMan5A

belongs to GH5 and displays exo-b-mannosidase activity,

releasing mannose from the nonreducing end of manno-

oligosaccharides and polysaccharides (Dias et al., 2004).

Analysis of the primary structures of CmEpiA and CmUnkA,

suggests that both proteins are nonmodular and intracellular.

Comparison of the sequence of the two deduced polypep-

tides with biological databanks showed that the protein

encoded by unkA has various protein homologues with no

demonstrated enzymatic function, while CmEpiA exhibits

55% sequence identity with a protein from M. degradans

(ZP_0315883) and lower identity scores (below 25%) with

bacterial N-acyl-D-glucosamine-2-epimerases and phospho-

mannose isomerases. In this work we were unable to assign a

biochemical role for CmUnkA.

Biochemical properties of CmAga27A

To investigate the biochemical properties of CmAga27A, the

protein was expressed in E. coli Origami and purified to

unkA man5AepiA aga27A

T TP P

−35 −10 Start

P TTAACT <21 bp> TATAAG <4 bp> A

P TTGACG <25 bp> TATAAT <5 bp> A

1 kb

Pst I Pst IEcoR I EcoR I

pZC2pZC3

pZC4

(a)

(b)

(c)

Fig. 1. Organization of the Cellvibrio gene cluster encoding galacto-

mannan degrading enzymes. Plasmids containing DNA fragments that

allowed the assembly of the 7-kb sequence containing the four genes

identified in this work are displayed in (a). Putative terminator and

promoter sequences identified in the sequence and the position and

orientation of the ORFs are displayed in (b). (c) depicts the sequences of

the putative promoters. P, promoter; T, terminator.

FEMS Microbiol Lett 261 (2006) 123–132c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

126 M.S.J. Centeno et al.

SaNP822650 TPPMGFNNWNSTGCRPEFNED--------MVKGTADIFVEKGLKDAGYQYVNLDDCWAL- 51SeAAC99325 TPPMGWNSWNSFGC--DIDER--------LIRDTADALVGSGMRDAGYQYVVVDDCWFD- 49CjBAB83765 TPPMGWNSWNIFGG--DINED--------KIKEIADAMVTTGMKDAGYEYVNLDDNWMAN 50CmAga27A TPQLGWNSWNTFAC--DVNEK--------MIREMADAMVASGMKDAGYEYINIDDCWHG- 49CjAga27A TPQMGWNSWNTFGC--NVDEK--------MIRAMADAMVTSGMKAAGYEYINIDDCWHG- 49MdZP00066516 TPPMGWNSWNNFGC--DVDEK--------LIKETADYMVSSGMKDAGYEYVNIDDCWHG- 49HsAAH02689 TPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMA- 59

SaNP822650 PARDSNGKLVPDPARFPGGIKAVADYVHSKGLKLGIYTSAGTKTCNEAG--FP---GALG 106SeAAC99325 PQRDPQGNLRANPERFPSGIRALADYVHSRGLKFGIYQVPTEKTCAQRGGTYPGATGSLG 109CjBAB83765 PARDANGKLIPDPKRFPSGMKALADYIHSKGLKFGIYGDRGVTTCCNIP-----QSGSQG 105CmAga27A -ERDKQGFIQVDKKSFPSGMKALADYVHSKGLKLGIYSDAGNTTCAGRP-------GSRG 101CjAga27A -ERDKNGFIQADKKHFPSGMKALADYVHAKGLKLGIYSDAGNTTCAGRP-------GSRG 101MdZP00066516 -ERDANGFIQADPERFPSGIKALADYVHSKGLKFGIYSDAGWTTCGGKP-------GSRG 101HsAAH02689 PQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFP-------GSFG 112

SaNP822650 HEYSDAQQFADWGVDYLKYDNCNNQG--VDAKLRYTTMRDALKATGRPIVYSLCEWG--- 161SeAAC99325 HEEQDARTFAEWGVDYLKYDWCSPEGTLEDQIAAFTKMRDALAATGRPIVYSINSNSYHP 169CjBAB83765 YEEQDAKTFAEWGLDYLKYDNCASDS---NLQAGYEKMRDALLKTGRPIFYSICCWY--- 159CmAga27A HEYQDAVTYASWGIDYVKYDWCDTKD--INPKAAYATMRDAIHKAGRPMLFSICEWG--- 156CjAga27A HEYQDALTYASWGIDYVKYDWCDTQD--INPKSAYATMRDAIHKAGRPMLFSICEWG--- 156MdZP00066516 YEFQDAQMYAKWGVDYLKYDWCATDG--LKAEGAYQTMREAIHKAGRPMVFSICEWG--- 156HsAAH02689 YYDIDAQTFADWGVDLLKFDGCYCDS-LENLADGYKHMSLALNRTGRSIVYS-CEWPLYM 170

SaNP822650 ---ENKPWEWASDVGQLWRTTGDISDSWGS-----------MLSILKQ----NLPLAPYA 203SeAAC99325 D--KNGATHDWSPVANMWRTTEDIKPVWDSGNEN--EYPMGVVNIIDV----NRGLAAQA 221CjBAB83765 -----FAGPWMVDCGNSWRTTGDISDSWGS-----------IIRNIDE----NSKSAAYA 199CmAga27A ---DNKPWEWATDVGHSWRTTGDIYPCWNCEHNHGSWSSWGVLPILDK----QAGLRKYA 209CjAga27A ---DNQPWEWAQDVGHSWRTTGDIYPCSNCEHNHGSWSSFGVLPILDK----QAGLRKYA 209MdZP00066516 ---DNQPWEWAKPIGHLWRTTGDIYNCFDCEYDHGTWSSWGVLQILDM----QDDLRQYA 209HsAAH02689 WPFQKPNYTEIRQYCNHWRNFADIDDSWKS-----------IKSILDWTSFNQERIVDVA 219

SaNP822650 GPGHWNDPDMLEVGN------SGMTDTEYRTHFSMWSIMAAPLLIGSDLRKASAATFDIL 257SeAAC99325 RPGHWNDPDMLEVGVYDVEGFKGLTDTEARAHLSMWALMASPLIAGNNVTRMPDGIRDIL 281CjBAB83765 GPGHWNDPDMLEVGN------GNMTDTEYKAHFSMWCMMAAPLIAGNDLRNMTPATKEIL 253CmAga27A GPGHWNDMDMMEVGN-------GMNEDEDRAHFSLWAMMASPLIAGNDLRKMSEATKKIL 262CjAga27A GPGHWNDMDMMECFN-------GMTEEEDRAHFSLWAFMASPLIAGNDLRNMSDTTRAIL 262MdZP00066516 GPGHWNDPDMMEVGN-------GMTEAEDRSHFSMWAMLAAPLIAGNDIRNMSEATRKIL 262HsAAH02689 GPGGWNDPDMLVIGN------FGLSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALL 273

SaNP822650 DNKEVIAVDQDPLGKQGTVLSSEGGRWAVTKEMK-DGS-RAVALFNETDSAQRITTTAQA 315SeAAC99325 TNREVVAVDQDPAGAQGVPVRDHGDREVWVKNMA-DGS-RVVALFNRGERPAGIRTTARE 339CjBAB83765 TNKEVIAIDQDAAGVQGTKVSSSGELEVWCKPLGTDGTTKAVALLNRGATSADITVNWSD 313CmAga27A TNKDMLAINQDKLGIQAMKWIDEGDIEIYVKPLE-KGD-YAVLFLNRADTTVNYSLDWGF 320CjAga27A THKETIAINQDKLGIQAMKWIDEGDLEIYIKPLE-KGH-YAVLFLNRADDAMDYRFDWSF 320MdZP00066516 TNKAVIAVDQDELGVQGFKYSSKNGVEVWFKPLA-NDE-WAMAVLNRNKGEVKFEFKWRN 320HsAAH02689 QDKDVIAINQDPLGKQGYQLRQGDNFEVWERPLS--GLAWAVAMINRQEIGGPRSYTIAV 331

SaNP822650 VGLPKAH----------AYTLRDLWQHRSYNTAGT-ISATVPAHGTVLVRVSA------- 357SeAAC99325 AGLPKAS----------EYQVRDLWAHETSTTDGE-IRAEVPAHGVVLLRVSAQG----- 383CjBAB83765 IQLADG-----------PVTVRDLWEHKDCGTFNTGYTANVPSHGVVVLKV--------- 353CmAga27A HYMKDDISKHEIFFDKKKFNWRDIWN-GGKGSTAEKLNLTMAAHSVAVLRLTPQ------ 373CjAga27A HYMKDDISKHEIFFDKQAFNWRNIWN-GETGSTKEVLNIKVPAHGVVVLRLSPR------ 373MdZP00066516 EVVKDELTHRTITFNEQKFDWQDLWNKSNKGHTKKFLKTKIAGHDTLMFRLTPAKN---- 376HsAAH02689 ASLGKGVACN---PACFITQLLPVKRKLGFYEWTSRLRSHINPTGTVLLQLENTMQMSLK 388

(a)

Fig. 2. Alignment of bacterial GH27 enzymes (a) and unrooted phylogenetic tree of functional a-galactosidases from GH27 (b). In (a), GH27 bacterial

a-galactosidases are aligned with the Homo sapiens homologue from which the 3D structure is known (Garman & Garboczi, 2004). Catalytic residues

(D152 and D207) and the tryptophan that stacks galactose at the active site (W40) are depicted in gray. The substrate recognition loop is boxed (see

text). Secondary structure elements are depicted using arrows (b-strands) and cylinders (a-helix). In (b), the scale bar indicates the number of

substitutions per position following alignment with CLUSTALW (Thompson et al., 1994) and bootstrap analysis using the same software. The tree was

displayed with TreeView (Page, 1996). Branches not circled contain sequences from fungi. Two letters that indicate the genus and species, respectively,

of the endogenous host precede the protein accession numbers (GenBankTM or Swiss-Prot).

FEMS Microbiol Lett 261 (2006) 123–132 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

127Galactomannan utilization by Cellvibrio mixtus

homogeneity by metal affinity chromatography (Fig. 3a).

The pH and temperature profiles of CmAga27A were

determined with pNPaGAL (Figs 4a and b). The enzyme

was found to display maximum catalytic activity at 50 1C

although it was rapidly inactivated at temperatures above

55 1C (not shown). The pH optimum of CmAga27A was

found to be of 8.5 and the enzyme, in common with the

majority of the extracellular plant cell wall hydrolases, was

completely resistant to proteolytic inactivation. Purified

CmAga27A display high levels of activity against pNPaGAL

although substrate inhibition was detected at substrate

concentrations above 20 mM. Enzyme kinetics were ob-

served over the range 0.01–10 mM pNPaGAL, allowing for

the estimation of the apparent kinetic parameters that gave

values of 184.9� 4.8 min�1 and 0.23� 0.02 mM for kcat and

Km, respectively. In contrast, the enzyme does not hydrolyse

aryl-b-mannosides, 4-nitrophenyl-b-glucopyranoside or

aryl-b-arabinosides or the polysaccharides xylan, hydro-

xyethyl and carboxymethyl cellulose, mannan, undecorated

glucomannan, lichenan and laminarin. Interestingly, CmA-

ga27A exhibited activity against carob, locust bean and guar

gum galactomannans. The kinetic constants displayed by the

enzyme against these substrates are presented in Table 2. The

capacity of CmAga27A to hydrolyse the a-1,6-galactoside

linkage in oligosaccharides of the raffinose family was

evaluated. The data demonstrated that the Cellvibrio mixtus

a-galactosidase display a very marginal capacity to hydrolyse

raffinose (0.01 U mg�1) and estaquiose (0.006 U mg�1).

Taken together the results suggest that CmAga27A is an

enzyme that Cellvibrio mixtus uses to remove galactose side

chains from galactomannans.

Biochemical properties of CmEpiA

Initially, the possibility that CmEpiA was the phosphoman-

nose isomerase responsible for the interconversion of man-

nose 6-phosphate into fructose 6-phosphate in Cellvibrio

mixtus, was evaluated. In the genetic locus identified in this

0.1

HjCAA93244

PpBAA22992

AnCAB46229

PsCAA08915

MvBAA33931

MvAAB35252

PcAAG24510PcAAG24511ZcAAA35280

ZmBAA99555TdBAA86883

SpCAA64759

ScAAA34770ScCAA85739

SpAAA34769

SmCAA64760

HsAAH02689

MmAAA74453

MdZP00066516

CmAga27A

CjAga27A

CjBAB83765

SaNP822650

SeAAC99325

PsCAF34023

LeAAF04591

OsBAB12570

HaBAC66445

CsABC88435

CtCAA32772

GmAAA73963PvAAA73964

CaCAI47559CaAAA33022

CcCAI47560

Bacteria

Mammals

Plants

Fungi

(b)

Fig. 2. Continued.

FEMS Microbiol Lett 261 (2006) 123–132c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

128 M.S.J. Centeno et al.

report, the gene encoding CmEpiA is located 50 to the gene

encoding CmMan5A which releases mannose from oligo-

and polysaccharides (Dias et al., 2004). This observation and

the considerable homology established between the enzyme

and other phosphomannose isomerases (see above) sup-

ports the proposed function of CmEpiA. The assay consisted

of a coupled reaction with two enzymes and used mannose

as the substrate. The data revealed that purified CmEpiA

(Fig. 3b) expresses no phosphomannose activity. Under the

same conditions the assay correctly determined the activity

of a commercial phosphmannose isomerase, demonstrating

that the enzyme system used was valid (results not shown).

In addition, CmEpiA was unable to convert mannose into

mannose 6-phosphate, suggesting that the enzyme also does

not exhibit mannokinase activity. Finally, the ability of

CmEpiA to function as an epimerase was evaluated. The

data revealed that CmEpiA displays epimerase activity,

converting mannose into glucose. No epimerase activity

was detected on N-acyl-glucosamine or mannose 6-phos-

phate (not shown). The optimum pH of the enzyme is 7.5

and the temperature at which it displays maximum catalytic

activity is 40 1C (Figs 4a and b). The kinetic parameters of

CmEpiA were determined at 37 1C, revealing values of

11.2� 0.3 min�1 and 53.5� 4.9 mM for kcat and Km, respec-

tively, during the conversion of mannose to glucose

(Fig. 4c). The activity of the epimerase is not affected by

the presence of EDTA and various cations, such as Ca21,

Mr (kDa)

97.0

66.0

43.0

30.0

20.1

14.4

1 2 3 Mr

Mr (kDa) Mr

97.0

66.0

43.0

30.0

20.1

2 3

(a)

(b)

Fig. 3. Hyperexpression and purification of recombinant CmAga27A

(a) and CmEpiA (b). BL21 cells were transformed with the respective

recombinant plasmids and induced with isopropyl-b-D-thiogalactopyra-

noside as described in Materials and methods. Soluble extracts from

uninduced (lane 1) and induced (lane 2) cells were separated by sodium

dodecyl sulfate polyacrylamide gel electrophoresis and the proteins were

purified through affinity chromatography (lane 3).

5 6 7 8 9 100

25

50

75

100

125

pH

Rel

ativ

e ac

tivity

(%

)

0 10 20 30 40 50 60 700

25

50

75

100

125

Temperature (°C)R

elat

ive

activ

ity (

%)

0 100 200 300 400 500 6000.0

2.5

5.0

7.5

10.0

12.5

Mannose (mM)

Glu

cose

(m

in−1

)

(a)

(b)

(c)

Fig. 4. Activity of CmAga27A (m) and CmEpiA (’) at different pH

values (a), temperatures (b) and kinetics of CmEpiA (c). Enzyme activity in

the Y-axis of (c) is expressed in molecules of glucose produced per

molecule of CmEpiA per minute.

FEMS Microbiol Lett 261 (2006) 123–132 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

129Galactomannan utilization by Cellvibrio mixtus

Zn21, Na1, Mg21, K1 and Mn21, suggesting that the

enzyme does not have any metal ion requirement.

Discussion

This report describes the cloning and characterization of a

Cellvibrio mixtus gene cluster encoding enzymes involved in

the degradation of galactomannan and in the metabolism of

its hydrolysis products. It is interesting to note that three of

the four genes identified are very tightly linked and encode,

respectively, a protein of unknown function, a mannose

epimerase (CmEpiA) and an exo-b-mannosidase (CmMa-

n5A). Recently we showed that CmMan5A is a cell

bound family 5 GH that releases mannose from the non-

reducing end of both oligo- and polysaccharides (Dias et al.,

2004). The fourth gene identified here encodes a family

27 a-galactosidase, termed CmAga27A, that is located at the

30 end of, and in the same orientation to, the other three

genes.

Based on substrate specificities, a-galactosidases have

been classified into two groups (Dey et al., 1993). One

group primarily removes galactose from oligosaccharides

such as melibiose, raffinose and staquiose. These enzymes

have potential biotechnological application in reducing the

detrimental effects associated with the ingestion of galactose

containing oligosaccharides by monogastric animals (Jin-

dou et al., 2002). These oligosaccharide specific enzymes

are located in GH families 4 and 36. The second group of

a-galactosidases acts preferentially on large macromole-

cules, such as galactomannans, glycoproteins and glycoli-

pids, while retaining the capacity to hydrolyse low-Mr

substrates. These enzymes are exclusive to GH family

27 (Garman & Garboczi, 2004). The biochemical properties

of CmAga27A, a family 27 GH highly homologous to the

eukaryotic enzymes, is unusual; the enzyme attacks galacto-

mannan but not oligosaccharides containing terminal

a-D-galactose residues. Similarly, the homologous a-galacto-

sidase from Clostridium josui, Aga27A, was also shown

to prefer galactomannan rather than small substrates (Jindou

et al., 2002). It is unknown if this property is shared by the

other enzymes of the bacterial cluster of GH27, although

Cellvibrio japonicus a-galactosidase CjAga27A displayed

some activity against raffinose and stachyose (Halstead

et al., 2000).

The substrate specificity of CmAga27A might reflect the

scarcity of oligosaccharides of the raffinose family on the

milieu of Cellvibrio mixtus. The presence of a typical signal

peptide in the primary sequence of CmAga27A suggests that

this enzyme is extracellular. This is consistent with the

complete resistance of the enzyme to proteolytic inactiva-

tion, a property typical of most extra-cellular plant cell wall

polysaccharidases that must retain stability within an highly

proteolytic environment (Fontes et al., 1995). Taken to-

gether the data suggest that CmAga27A acts cooperatively

with the extracellular repertoire of endo-mannanses releas-

ing galactose and manno-oligosaccharides from galacto-

mannans, which are then absorbed by Cellvibrio mixtus.

Interestingly, CmAga27A does not possess an associated

carbohydrate binding module typical of GH acting on

complex polysaccharidases. Analysis of the molecular archi-

tectures of family 27 enzymes demonstrates that none of the

enzymes display a modular architecture, suggesting that

removal of galactose from complex macromolecules does

not require the presence of carbohydrate binding modules.

Ultimately, this might reflect the high solubility presented

by the decorated galactomannans and/or the relative acces-

sibility of the galactose side chains in the complex poly-

saccharide.

In contrast to CmAga27A, the lack of a signal peptide in

the primary sequence of CmEpiA might reflect its intracel-

lular location. It is generally assumed that mannose meta-

bolism in bacteria follows its phosphorylation and transport

into the cytoplasm by a transmembrane sugar kinase/

transporter with the subsequent isomerization to fructose

6-phosphate through the action of a phosphomanose iso-

merase. The data presented here suggest that an alternative

metabolic pathway for the utilization of mannose might

exist in Cellvibrio mixtus. Intracellular nonphosphorylated

mannose molecules can be directly converted into glucose

through the action of CmEpiA. It remains to be established

Table 2. Kinetic parameters of CmAga27A during the hydrolysis of pNPaGAL and various galactomannans

Substrate kcat (min�1)� Km (g L�1) kcat/Kmw

pNPaGAL 184.9� 4.8 0.23� 0.02z 803.91

Guar gum galactomannan 5.26� 0.5 0.82� 0.06 6.42

Locust bean galactomannan 8.27� 0.8 1.10� 0.07 7.52

Carob galactomannan (hv) 8.81� 0.4 0.62� 0.03 14.21

Carob galactomannan (lv) 31.3� 0.9 2.21� 0.04 14.16

�kcat are in molecules of product per molecule of enzyme per minute.wkcat/Km values are in (g L)�1 min�1.zSubstrate concentration in mM.

pNPaGAL, p-nitrophenyl a-D-galactopyranoside; hv, high viscosity; lv, low viscosity.

FEMS Microbiol Lett 261 (2006) 123–132c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

130 M.S.J. Centeno et al.

whether both metabolic pathways coexist in Cellvibrio

mixtus and, if so, which one predominates. As genes encod-

ing CmEpiA and CmMan5A are tightly linked, it is tempting

to speculate that the product generated by CmMan5A,

mannose, serves as substrate for CmEpiA. This observation

suggests that CmEpiA might be a fundamental enzyme in

the metabolism of mannose in aerobic soil bacteria. Indeed,

support for this view is provided by the genome sequence of

Cellvibrio japonicus, which reveals an identical gene arrange-

ment to Cellvibrio mixtus within the manA locus. Cellvibrio

japonicus contains a similar tight cluster of three genes

encoding CjUnkA, CjEpiA and CjMan5A, which display

86%, 73% and 54% sequence identity with their Cellvibrio

mixtus counterparts. Downstream of this locus is the gene

encoding CjAga27A, which exhibits 83% sequence identity

with the Cellvibrio mixtus a-galactosidase. Although it

might be tempting to speculate that CjUnkA and CmUnkA

mediate transport of mannose across the inner membrane,

this appears unlikely as the proteins display no membrane-

spanning motifs. Interestingly, however, immediately up-

stream of the Cellvibrio japonicus man5A-containing genetic

cluster, is a gene whose product displays significant sequence

identity to sodium-dependent sugar transporters, and thus

may mediate the trafficking of mannose across the inner

membrane. Cellvibrio japonicus also contains genes encod-

ing the three key enzymes of the Leloir pathway, galacto-

kinase (galK), galactose 1-phosphate uridylyltransferase

(galT) and UDP-galactose 4-epimerase (galE), which cata-

lyse the conversion of galactose to the more metabolically

useful sugar glucose 1-phosphate. Galactokinase and galac-

tose 1-phosphate uridylyltransferase are encoded by a two-

gene operon, while galE is not linked to galK or galT. The

genes encoding the Leloir pathway are not linked to the

man5A locus. As Cellvibrio mixtus and Cellvibrio japonicus

are able to release galactose and mannose from galactoman-

nan through the same complement of enzyme activities, it is

highly likely that both Cellvibrio strains will also contain the

genes encoding the Leloir pathway.

Collectively, the data presented here and in other reports

suggest that galactomannan hydrolysis by Cellvibrio results

from the synergistic interaction of a large repertoire of

enzymes. Family 5 mannanases and CmAga27A are secreted

into the extracellular milieu where the mannose-containing

hemicelluloses are located (Hogg et al., 2003). The action of

CmAga27A exposes the mannan backbone to modular

family 5 mannanases. Membrane bound family 26 man-

nanses and CmMan5A further degrade the generated

manno-oligosaccharides and soluble galactomannans into

mannose and mannobiose (Halstead et al., 2000). These

hydrolysis products, which are generated at the surface of

the organism, can be readily taken up by Cellvibrio mixtus.

The absorbed mannose can be converted to glucose in the

cytoplasm through the action of CmEpiA.

Acknowledgements

This work was supported by grants SFRH/BD/16731/2004

(to C.I.P.D.G.), PraxisXXI/BD/21250/1999 (to F.M.V.D),

and POCI/CVT/61162/2004 from the Fundacao para a

Ciencia e a Tecnologia, Portugal.

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