Báo cáo khoa học: LmbE proteins from Bacillus cereus are de-N-acetylases with broad substrate specificity and are highly similar to proteins in Bacillus anthracis pot

Kinetic analysis of the activity of BC1534 and BC3461 on GlcNAc and GlcNAc2 revealed that GlcNAc2 is the favored substrate for both native enzymes.. Enzymatic properties of BC1534 and BC

with broad substrate specificity and are highly similar

to proteins in Bacillus anthracis

Alexandra Deli1, Dimitrios Koutsioulis2, Vasiliki E Fadouloglou2,*, Panagiota Spiliotopoulou1, Stavroula Balomenou1, Sofia Arnaouteli1, Maria Tzanodaskalaki2, Konstantinos Mavromatis3, Michalis Kokkinidis1,2and Vassilis Bouriotis1,2

1 Department of Biology, Enzyme Biotechnology Group, University of Crete, Greece

2 Institute of Molecular Biology and Biotechnology, Heraklion, Crete, Greece

3 Department of Energy ⁄ Joint Genome Institute, Genome Biology Program, Walnut Creek, CA, USA

Keywords

Bacillus anthracis; de-N-acetylase;

glucosamine; LmbE; mutational analysis

Correspondence

V Bouriotis, Department of Biology,

Enzyme Biotechnology Group, University of

Crete, PO Box 2208, Vasilika Vouton

714 09, Heraklion, Crete, Greece

Fax: +30 2810 394055

Tel: +30 2810 394375

E-mail: bouriotis@imbb.forth.gr

*Present address

Department of Biochemistry, University of

Cambridge, UK

(Received 22 October 2009, revised 15

March 2010, accepted 20 April 2010)

doi:10.1111/j.1742-4658.2010.07691.x

The genomes of Bacillus cereus and its closest relative Bacillus anthracis each contain two LmbE protein family homologs: BC1534 (BA1557) and BC3461 (BA3524) Only a few members of this family have been biochemi-cally characterized including N-acetylglucosaminylphosphatidyl inositol (GlcNAc-PI), 1-d-myo-inosityl-2-acetamido-2-deoxy-a-d-glucopyranoside (GlcNAc-Ins), N,N¢-diacetylchitobiose (GlcNAc2) and lipoglycopeptide antibiotic de-N-acetylases All these enzymes share a common feature in that they de-N-acetylate the N-acetyl-d-glucosamine (GlcNAc) moiety of their substrates The bc1534 gene has previously been cloned and expressed

in Escherichia coli The recombinant enzyme was purified and its 3D struc-ture determined In this study, the bc3461 gene from B cereus ATCC14579 was cloned and expressed in E coli The recombinant enzymes BC1534 (EC 3.5.1.-) and BC3461 were biochemically characterized The enzymes have different molecular masses, pH and temperature optima and broad sub-strate specificity, de-N-acetylating GlcNAc and N-acetylchito-oligomers (GlcNAc2, GlcNAc3 and GlcNAc4), as well as GlcNAc-1P, N-acetyl-d-glu-cosamine-1 phosphate; GlcNAc-6P, N-acetyl-d-glucosamine-6 phosphate; GalNAc, N-acetyl-d-galactosamine; ManNAc, N-acetyl-d-mannosamine; UDP-GlcNAc, uridine 5¢-diphosphate N-acetyl-d-glucosamine However, the enzymes were not active on radiolabeled glycol chitin, peptidoglycan from B cereus,

N-acetyl-d-glucosaminyl-(b-1,4)-N-acetylmuramyl-l-alanyl-d-isoglutamine (GMDP) or N-acetyl-d-GlcN-Na1-6-d-myo-inositol-1-HPO4 -octadecyl (GlcNAc-I-P-C18) Kinetic analysis of the activity of BC1534 and BC3461 on GlcNAc and GlcNAc2 revealed that GlcNAc2 is the favored substrate for both native enzymes Based on the recently determined crystal structure of BC1534, a mutational analysis identified functional key resi-dues, highlighting their importance for the catalytic mechanism and the sub-strate specificity of the enzyme The catalytic efficiencies of BC1534 variants were significantly decreased compared to the native enzyme An alignment-based tree places both de-N-acetylases in functional categories that are dif-ferent from those of other LmbE proteins

Abbreviations

GalNAc, N-acetyl- D -galactosamine; GlcNAc, N-acetyl- D -glucosamine; GlcNAc 2 , N,N ¢-diacetylchitobiose; GlcNAc-1P, N-acetyl- D -glucosamine-1 phosphate; GlcNAc-6P, N-acetyl- D -glucosamine-6 phosphate; GlcNAc-Ins, 1- D -myo-inosityl-2-acetamido-2-deoxy-a- D -glucopyranoside; GlcNAc-PI, N-acetylglucosaminylphosphatidyl inositol; GlcNAc-I-P-C18, N-acetyl- D -GlcN-a1-6- D -myo-inositol-1-HPO4-octadecyl; GMDP, N-acetyl- D -glucosaminyl-(b-1,4)-N-acetylmuramyl- L -alanyl- D -isoglutamine; ManNAc, N-acetyl- D -mannosamine; UDP-GlcNAc, uridine

5¢-diphosphate N-acetyl- D -glucosamine.

Bacillus cereus, an opportunistic pathogen that causes

food poisoning, and Bacillus anthracis, the

endospore-forming bacterium that causes inhalational anthrax,

share a large number of homologous genes, as

demon-strated by recent genome sequencing and comparative

analysis [1,2] Given the laboratory safety precautions

necessary for working with highly infectious agents

and the recent concerns regarding use of B anthracis

as a potential bioweapon (class A agent, Centers for

Disease Control), the B cereus enzymes are useful

models for studying the corresponding proteins of

B anthracis

The ERGO light database of the B cereus

ATCC14579 genome (http://www.ergo-light.com/)

reveals the presence of two LmbE protein family

homologs, BC1534 and BC3461, which share 21%

identity [1] Furthermore, they share 96% and 95%

identity with their homologs BA1557 and BA3524,

respectively, from B anthracis The LmbE protein

family (Fig 1) includes

N-acetylglucosaminylphosphat-idyl inositol (GlcNAc-PI) de-N-acetylases from

mammals [3], yeast [4] and protozoa [5],

1-d-myo-inosi-tyl-2-acetamido-2-deoxy-a-d-glucopynanoside

(GlcNAc-Ins) de-N-acetylase from Mycobacterium tuberculosis

(MshB) (EC 3.5.1.89) [6], N,N¢-diacetylchitobiose

(GlcNAc2) de-N-acetylase from the archaeon

Thermo-coccus kodakaraensis KOD1 (Tk-Dac) (EC 3.5.1.-) [7]

and antibiotic de-N-acetylases from Actinoplanes

teich-omyceticus (Orf2) [8] and Bacillus circulans (BtrD) [9]

The most important members of the LmbE protein

family, together with the structures of their substrates,

are shown in Fig 2

The crystal structure of BC1534 (previously reported

as BcZBP) has been determined at 1.8 A˚ resolution (Fig 3A) [10] The structures of three other LmbE protein family members have been similarly deter-mined, namely TT1542 from Thermus thermophilus [11], MshB from Mycobacterium tuberculosis [12] and Orf2 from Actinoplanes teichomyceticus [8] The N-terminal part of the 234 amino acid BC1534 protein adopts a Rossmann fold, and the C-terminal part con-sists of two b-strands and two a-helices In the crystal, the protein forms a compact hexamer (Fig 3A), in agreement with solution data [13] A zinc binding site and a potential active site have been identified in each monomer These sites have extensive similarities to those found in other known zinc-dependent hydrolases with de-N-acetylase activity, i.e MshB from Mycobac-terium tuberculosis [12] and LpxC (UDP-(3-O-(R-3-hy-droxymyristoyl))-N-acetylglucosamine de-N-acetylase) from Aquifex aeolicus [14] Despite a low degree of structural homology, it has been suggested that these enzymes are the products of convergent evolution due

to similar active site features

The objective of this study was to shed light on the role of two de-N-acetylases from B cereus, which are LmbE protein family homologs Given the exten-sive homology between B cereus and B anthracis, the results of these studies could contribute to understand-ing of the physiology of this interestunderstand-ing pathogenic microbe Two recombinant de-N-acetylases from

B cereus ATCC14579 were biochemically character-ized A comparison of the substrate specificities of the enzymes with those of other members of the LmbE

D

RHPDHA

VHPDHN

VHPDHD SGHSNH

GHPDHV

RHPDHT

GHPDHR

EHPDHE

KHVDHR

A ADDVEIGMAGTIAKYTKQG

D

QG´

PHPDDGELGCGGTLARAKAEG

PHFDDVILSCASTLMELMNQG

PHLDDAVLSFGAGLAQAAQDG PHPDDCAIGLGGTIKKLTDSG

AHPDDESLSNGATIAHYTSRG

AHPDDEAMFFAPTILGLARLK

A ADDVEIGMAGTIAKYTKQG

114 126

126 114

157

152 165 167

148 112

31 30

30 31

68

59 35 33

32 29

BC3461

BA1557 BA3524

PIG-L MshB TT1542

Orf2

BtrD

Tk-Dac

Fig 1 Partial amino acid sequence alignment of BC1534 and BC3461 with five LmbE-like proteins of known function and three of unknown function (BA1557, BA3524 and TT1542) BA1557, hypothetical protein from Bacillus anthracis strain Ames (NP_844007); BA3524, hypotheti-cal protein from Bacillus anthracis strain Ames (NP_845802); PIG-L, GlcNAc-PI de-N-acetylase from Rattus norvegicus (BAA20869); MshB, GlcNAc-Ins de-N-acetylase from Mycobacterium tuberculosis H37Rv (NP_215686); TT1542, conserved hypothetical protein from Ther-mus thermophilus HB8 (BAC67240); Tk-Dac, N,N¢-diacetylchitobiose de-N-acetylase from Thermococcus kodakaraensis KOD1 (BAD29713); Orf2, de-N-acetylase from Actinoplanes teichomyceticus (CAG15014); BtrD, de-N-acetylase from Bacillus circulans (BAE07068) The black regions indicate identical residues and gray shading indicates similar amino acids.

protein family is presented A mutational analysis of

BC1534 identified functional key residues, highlighting

their importance for the catalytic mechanism and the

substrate specificity of the enzyme A computational

analysis to predict the biological role of the enzymes is

also reported

Results

Identification of bc1534 and bc3461 from

B cereus ATCC14579 and comparison with

B anthracis homologs

In silico analysis of the B cereus ATCC14579 genome

revealed the presence of the bc1534 and bc3461 genes

(NP_831313 and NP_833195, respectively) The bc1534

gene consists of 705 bp and encodes a protein of 234

amino acids, while the bc3461 gene consists of 663 bp

and encodes a protein of 220 amino acids Neither

N-terminal signal sequences nor transmembrane helices

were found in the deduced amino acid sequences (based

on sequence similarities and the sequence prediction

programs http://www.cbs.dtu.dk/services/SignalP/ and

http://www.sbc.su.se/~miklos/DAS/ for signal peptide and transmembrane domain prediction, respectively), which is consistent with the fact that recombinant BC1534 and BC3461 were detected and purified from the cytosolic fraction of Escherichia coli cells as intra-cellular enzymes

According to the Pfam [15] and Cluster of Ortholo-gous Groups [16] databases, BC1534 and BC3461 are members of the LmbE protein family (Pfam02585⁄ COG2120) BC1534 is also classified as a member of carbohydrate esterase family 14 (CE14), but BC3461 has not been assigned to any of the CE families Two open reading frames found in B anthracis strain ‘Ames ancestor’ [BA1557 (NP_844007) and BA3524 (NP_845802)] were identical in size and shared identities

of 96% and 95% to BC1534 and BC3461, respectively The bc1534 gene belongs to an operon that also con-tains six genes that have various predicted functions (Fig 4) In silico data show that expression of the operon is regulated by a common mechanism (the rE promoter at the 5¢ end of the operon) [17] Based on the gene organization and predicted functions of the genes that belong to this operon, there is no apparent

GlcNAc GlcNAc2

OH

OH O O

CH 3

CH2OH

H 3 C

NH HO

HO

HO HO

HO O O

2

NH2

HO NH

HO

HO

OH

OH

OH OH N

OH OH OH

OH OH HO

HO

HO

CH3

CH 3

H3C

OHOH

OH HO

OH

OH OH NH

O O

O O O

O ′ O′

P

HN

Cl

Cl H

N

N HN H

N

HO

O

O O O O O O

O

O O O O

O O

O

O

HO

HO

HO 3

1

HO HO H

GlcNAc-Ins

BtrD

N-acetyl-D -glucosaminyl

aglycone

N-acetyl-D -glucosaminyl pseudoaglycone

Orf2

GlcNAc-PI

PIG-L

Fig 2 LmbE proteins substrates All enzymes catalyze hydrolysis of the N-acetyl group (shown inside a circle or a rectangle) of the GlcNAc moiety of their substrate(s).

A B

Fig 3 (A) Overall structure of BC1534 hexamer formed through the association of three dimers [10] (PDB ID 2ixd) Dimerization is achieved through interaction between the b8-strand of one monomer and the b6- and b7-strands of the other monomer (B) Surface representation of the enzyme and view of the active site from outside The active site is occupied by GlcNAc 2 (shown as a ball model) The substrate has been modeled into the active site by autodocking The two conformations of R140, determined by the crystal structure, are also shown (stick models) (C,D) GlcNAc2(stick model) has been docked into the active site of the enzyme, and the interactions or loss of interactions with residues that occupy positions 140 and 42 are indicated (C) Position 140 The native Arg residue (carbon atoms in yellow) and the mutations Ala (carbon atoms in brown) and Glu (carbon atoms in gray) are shown as stick models The distances between the substrate and each of the three residues are shown as dashed lines (D) Position 42 The native Ala residue (yellow) and the mutation Ser (gray) are shown

as stick models The positions of the two native Ser residues (45 and 46) in the neighborhood of the mutation are shown as thin stick mod-els The distances from the substrate are shown as dashed lines All the point mutations shown in (C) and (D) were computationally intro-duced in the model of the native crystal structure (see Experimental procedures) (E) Cartoon representation of the active site of BC1534, highlighting residues that have been mutated and showing their relative positions in the structure The H110 ⁄ D112 pair is shown as a stick model, and three of the residues that form a hydrophilic pocket suggested to function as the ‘oxyanion hole’ are shown using Van der Waals dotted spheres (F) Cartoon representation that focuses on the entrance of the active site tunnel, showing that it is dominated by positively charged residues represented here by Van der Waals dotted spheres The position of K207 is also shown.

pathway in which all of these genes could be involved.

In contrast, bc3461 is not part of a gene cluster,

indi-cating that its expression is probably regulated in an

independent manner (Fig 4)

Protein sequence alignment of characterized

mem-bers of this family with the B cereus homologs (Fig 1)

revealed two conserved sequence motifs The first

[(A⁄ P)H(X ⁄ P)DD] is located near the N-terminus, and

the second [H(X⁄ P)DH] is located towards the middle

of the protein The crystal structure of BC1534 [10]

revealed that the underlined H and D residues in the

first motif and the last H of the second motif are zinc

binding ligands Moreover, it has been proposed for

other members of the family that the underlined H

and the subsequent D of the second motif play a

charge relay role during catalysis [9]

Cloning, expression and purification of BC1534

The gene encoding BC1534 was isolated from B cereus

ATCC14579 genomic DNA by PCR and cloned into

expression vector pET26b for recombinant protein

production in E coli BC1534 was produced as a

C-terminal hexahistidine fusion protein to facilitate

puri-fication by affinity chromatography (Ni-nitrilotriacetic

acid) SDS⁄ PAGE analysis revealed the presence of

three protein bands The position of the main band is

in agreement with the predicted molecular mass of the

hexahistidine fusion protein (27 kDa) The N-terminal

amino acid sequence of the protein bands

correspond-ing to the higher-molecular-mass proteins seen in

SDS⁄ PAGE was determined to be MSGL, which is identical to the predicted amino acid sequence of BC1534 (MSGLHILAFG), suggesting the existence of non-denatured homopolymers of BC1534 in the SDS⁄ PAGE gel (Fig 5A) The positions of these bands are in agreement with the molecular mass of purified BC1534 estimated by size-exclusion chroma-tography [13] and glutaraldehyde cross-linking [18] (approximately 160 kDa), indicating that the protein exists as a hexamer in solution

Cloning, expression and purification of BC3461 bc3461 was isolated from B cereus ATCC14579 geno-mic DNA by PCR and cloned into the pRSETA expression vector for recombinant protein production

in E coli Purification was achieved in two steps, using ion-exchange and size-exclusion chromatography The apparent molecular mass of BC3461 was calculated to

be 26 kDa as determined by SDS⁄ PAGE (Fig 5B) and gel-filtration chromatography, suggesting that the pro-tein exists as a monomer in solution

Enzymatic properties of BC1534 and BC3461 BC1534 and BC3461 were active on N-acetyl-d-gluco-samine (GlcNAc), N-acetylchitooligomers (GlcNAc2, GlcNAc3 and GlcNAc4), GlcNAc-1P, GlcNAc-6P, GalNAc and ManNAc (Table 1) The specificity of the enzymes for various N-acetylchito-oligomers was examined, and the kinetic parameters were determined

BC1531

BC1532

1482325

Putati

ve transcriptional regulatory pr

otein

Dih ydr odipicolinate reducatase

Short chain deh ydr

ogenase

LmbE-r elated

pr otein Hypothetical pr

otein

Hypothetical pr

otein

Arsenical pump membrane pr otein

LmbE-r elated

pr otein Glycosyltransferase

tRN

A CCA-pyr ophosphorylase Biotin-opemn repr

ess or/biotin–[acetyl-CoA-carboxylase]

synthetase Meth

ylgly oxal

synthase

BC3461

BC3462

BC3463

BC1537

1487879 A

B

Fig 4 Gene organization in the 5.5 kbp region that includes bc1534 (A) and the 4.3 kbp that includes bc3461 (B) on the B cereus ATCC14579 genome Arrows indicate open reading frames Genes of interest are indicated by colored arrows.

(Tables 1 and 2) BC1534 and BC3461 exhibited

maxi-mum activity towards GlcNAc2 Kinetic parameters

for GlcNAc and GlcNAc2were obtained from

Linewe-aver–Burk plot analysis, and the enzyme reaction rates

for these substrates appear to follow Michaelis–

Menten kinetics The resulting kcat⁄ Kmratios (catalytic efficiency, Keff) indicated that GlcNAc2 was the favored substrate for both BC1534 and BC3461 (Table 2) UDP-GlcNAc was also tested as a potential substrate due to the presence of a glycosyltransferase

104

175

47.5 62

25 32.5 83

16.5

BC3461

6.5

37

97 50

29 20

BC1534

Fig 5 SDS ⁄ PAGE of the purified LmbE-like

proteins BC1534 (A) and BC3461 (B) (A)

Lane 1, molecular weight markers; lane 2,

size-exclusion chromatography eluant.

Samples were electrophoresed on a 12%

polyacrylamide gel under denaturing

conditions Protein bands were visualized

by staining with Coomassie Brilliant Blue

R-250.

Table 2 Kinetic parameters of BC3461 and BC1534 (wild-type and variants) towards GlcNAc and GlcNAc2 NA, not active.

Enzyme

Substrate

kcat(s)1) Km(l M ) Keff(l M )1Æs)1) k

cat (s)1) Km(l M ) Keff(l M )1Æs)1)

Table 1 Substrate specificity (percentage relative activity)of BC3461, BC1534 and mutants Assay conditions were 25 m M HEPES ⁄ NaOH

pH 8.0, 200 m M NaCl, 1 m M CoCl2for 30 min at 37 C for BC1534 and its variants, and 25 m M MES ⁄ NaOH pH 6.5, 200 m M NaCl, 1 m M MgCl 2 for 30 min at 20 C for BC3461 The concentration of substrates was adjusted with respect to their content in terms of N-acetyl residues ND, not determined.

gene (bc1535) downstream BC1534 (Fig 4) The Km

value (3 lm) was comparable to that for GlcNAc2, but

the Keff value (0.02 lm)1Æs)1) was significantly lower

compared to that for GlcNAc2

The purified recombinant enzymes showed different

pH and temperature optima using GlcNAc2 as

sub-strate BC1534 exhibited a pH optimum at pH 8.0,

and optimum temperature for enzyme activity was

determined to be 37C The pH and temperature

optima for BC3461 were 6.5 and 20C, respectively

Moreover, a 3.5-fold increase in BC1534 activity was

observed on addition of 1 mm CoCl2 to the assay

buf-fer, and a twofold increase in BC3461 activity was seen

when 1 mm MgCl2 was added BC1534 and BC3461

were inhibited by the presence of 1 mm Cu2+ and

1 mm Zn2+(both tested as chlorides), similar to

previ-ous reports on zinc hydrolases [19] The enzymes were

not inhibited by acetate or EDTA even at

concentra-tions up to 50 and 20 mm, respectively Moreover, they

were both inactive on radiolabeled glycol chitin and

peptidoglycan from B cereus vegetative cell walls, and

BC1534 was also inactive on GlcNAc-I-P-C18, a

syn-thetic analogue of GlcNAc-PI

Mutational analysis of BC1534

In order to elucidate the importance of selected

resi-dues in the catalytic mechanism, substrate affinity and

specificity, as well as to investigate the importance of

the oligomerization state on the activity of the enzyme,

six point mutations and one fragment deletion were

performed The enzyme variants obtained were tested

against the same range of substrates as the wild-type

enzyme, and their substrate specificities and kinetic

parameters are presented in Tables 1 and 2

BC1534 shares extensive similarities to the active

sites of two characterized zinc-dependent deacetylases,

i.e MshB and LpxC [6,14] Based on these similarities,

it was proposed [10] that BC1534 would utilize a

simi-lar catalytic mechanism to these enzymes The

domi-nant features of this mechanism are an H⁄ D charge

relay pair (H110⁄ D112 for BC1534, Fig 3E) and an

‘oxyanion hole’ (mainly formed by the side chains of

Y194, N150 and D108 in BC1534, Fig 3E) To test

this hypothesis, we mutated D112 and Y194 to

resi-dues that lack groups with a putative role in catalysis

Thus, D112 was mutated to asparagine, which retains

all aspartate’s stereochemical and physicochemical

properties except its ability to relay protons The

mutant protein (D112N) was completely inactive on

most of the substrates tested, i.e GlcNAc, GlcNAc4,

GlcNAc5, GlcNAc6 and GlcNAc-1P, but exhibited

higher relative activity against GlcNAc-6P and

Man-NAc than the native enzyme did (Table 1) Y194 was mutated to phenylalanine, which retains all the hydro-phobic interactions of the initial residue but lacks the hydroxyl group that is proposed to participate in for-mation of the ‘oxyanion hole’ The Y194F mutant showed the broadest substrate specificity among BC1534 variants, but its catalytic efficiency against the preferred substrate of the native enzyme (GlcNAc2) was highly reduced In addition, it exhibited activity against GlcNAc5, GlcNAc6 and GMDP, in contrast to the wild-type enzyme, which was inactive with these substrates

Based on the crystal structure of BC1534 [10] and a molecular dynamics study [20], we proposed that the rim and the loops surrounding the active site tunnel could play a significant role in the substrate specificity

of the enzyme We experimentally tested this hypothe-sis by mutating R140, K207 and A42, three residues located on the rim of the active site tunnel In the crys-tal structure, R140 adopts two discrete conformations (Fig 3B) One of these conformations highly restricts the accessibility of the active site, while the other, which is mainly stabilized by electrostatic interactions with the adjacent E142 and backbone carbonyl groups, keeps the entrance open To further investigate the role

of R140, it was mutated (a) to a small hydrophobic residue, Ala and (b) to a negatively charged Glu resi-due With minor exceptions, both variants exhibited the same preferences as the native enzyme for the sub-strates tested (Table 1) R140A showed a significant decrease in efficiency compared to the native enzyme for GlcNAc2 and GlcNAc However, this variant exhibited 100 times higher catalytic activity (kcat) on GlcNAc than the wild-type enzyme In the case of R140E, the Km values remain similar to that of the native enzyme, but the kcat is significantly decreased for both substrates

K207 was mutated to isoleucine, a small hydropho-bic residue Remarkably, this variant was inactive on all substrates tested A42 is located in a position favor-able for the formation of hydrogen bonds with the substrate’s hydroxyl groups (Fig 3D) Thus, A42 was mutated to a serine, which is a residue of comparable size to alanine and its side chain has the ability to form hydrogen bonds Unexpectedly, the produced variant (A42S) exhibited a dramatic reduction in Keff value for both substrates (Table 2)

BC1534 is a hexamer (Fig 3A) that may be consid-ered as a trimer of dimers [10,20] Dimerization is mainly established via exchange of two short b-strands between the monomers (b8-strand in Fig 3A) Dele-tion of the b8-strand by PCR resulted in an insoluble and inactive protein

Prediction of function

Although members of Pfam02585 (LmbE protein

fam-ily) share common sequence features, they do not

exhi-bit the same function [5–8] Sequence alignment of 929

proteins belonging to Pfam02585 revealed a number of

different sequence groups (Fig 6), presumably

reflect-ing different functions All proteins for which the

enzy-matic function has been studied belong to different

groups The two Bacillus proteins were placed in

func-tional categories different from other LmbE proteins

As the sequence of the protein is not sufficient to

reveal the function of the enzyme, we sought other

lines of evidence to predict its function Location of

enzymes in the same chromosomal neighborhood could

indicate a functional relationship and frequently helps

to predict the function of enzymes [21] BC1534

appears to be in a chromosomal neighborhood that is

conserved among the Bacillus species (data not shown)

Microarrays [22] and deep sequencing of the

transcrip-tome [23] in B anthracis revealed that ba1557 (the

homolog of bc1534) is in an operon surrounded by

genes homologous to bc1531-bc1537, and is expressed

during the early and mid log phases, while ba3524 (the

homolog of bc3461) is expressed in the early log phase

and during sporulation Despite possible differences in

the life cycle of these Bacillus species, these data from

the B anthracis transcriptome [22,23] could provide

strong evidence for similar gene expression in B cereus

In other members of the Firmicutes, the conservation

is restricted to the presence of a glycosyltransferase

(Pfam00534) downstream of the LmbE-related protein (i.e the proteins encoded by the bc1535 and bc1534 genes, respectively, in B cereus)

Discussion

In an effort to shed light on the role of the LmbE pro-tein family enzymes in bacteria and contribute to the understanding of the pathobiology of B anthracis, we describe biochemical characterization of the recombi-nant enzymes BC1534 and BC3461 from B cereus BC1534 exhibited overall 21% identity and 31% simi-larity with BC3461 Moreover, BC1534 and BC3461 shared 96% and 95% identity with their homologs BA1557 and BA3524, respectively, from B anthracis (Fig 1) The purified recombinant enzymes exhibited different molecular masses (Fig 5), pH and temperature optima and were not inhibited by acetate or EDTA BC1534 and BC3461 were activated by CoCl2 and MgCl2, respectively Both enzymes were effective in de-N-acetylating GlcNAc and N-acetylchitooligomers (GlcNAc2, GlcNAc3and GlcNAc4), as well as GlcNAc-1P, GlcNAc-6P, GalNAc, ManNAc and UDP-GlcNAc (Table 1) However, the enzymes were not active on glycol chitin or peptidoglycan from B cereus ATCC14579, GMDP or GlcNAc-I-P-C18 Kinetic analysis of BC3461 and BC1534 towards the N-acetylchito-oligosaccharides GlcNAc and GlcNAc2 revealed that GlcNAc2 is the favored substrate for both enzymes Comparison of the Keff values showed that both enzymes are equally effective on GlcNAc BC1534 was six times more

THA1200 Ther mus ther mophilus HB8

BC3461 Bacillus cereus A

T

CC 14579

TK1764 Ther mococcus kodakar aensis KOD1

BC1534 Bacillus cereus A

TCC 14579

BtrD Bacillus circulans

Rv1170 Mycobacter

ium tuberculosis H37Rv

Fig 6 Neighbor joining tree for 929 LmbE

proteins Bacillus cereus proteins and

pro-teins of known function are indicated.

effective than BC3461 (Table 2) when GlcNAc2 was

tested as substrate

Chitin de-N-acetylases from fungi and insects [24],

chito-oligosaccharide de-N-acetylases from Rhizobium

(NodB) [25] and Vibrio parahaemolyticus [26], and

Glc-NAc peptidoglycan de-N-acetylases [27] are considered

to be catalytically similar de-N-acetylases They are all

members of carbohydrate esterase family 4, catalyzing

the hydrolysis of N-linked acetyl groups on GlcNAc

residues Chitin de-N-acetylase is capable of removing

N-acetyl groups from chitin chains [28] NodB, which

is involved in nodulation signal synthesis,

de-lates the non-reducing GlcNAc residue of

N-acety-lchito-oligosaccharides [25] GlcNAc peptidoglycan

de-N-acetylase increases the resistance of peptidoglycan

to lysozyme via de-N-acetylation of GlcNAc residues

[27] None of the above enzymes accepts GlcNAc as

substrate

The biochemically characterized LmbE proteins are

members of distinct metabolic pathways MshB is

involved in the second step of mycothiol biosynthesis

[6], whereas GlcNAc-PI de-N-acetylases play an

impor-tant role in biosynthesis of the

glycosylphosphatidyli-nositol biosynthesis in eukaryotes [3–5] Orf2 [8] and

BtrD [9] are involved in the synthesis of

lipoglycopep-tide antibiotics, while Tk-Dac plays an essential role in

a novel chitinolytic pathway identified in archaea [7]

All members of the LmbE protein family with known

function share a common feature in that they

de-N-acet-ylate the GlcNAc moiety of their substrates (Fig 2) It

has been reported that MshB [6], Orf2 [8] and PIG-L

(GlcNAc-PI de-N-acetylase from Rattus norvegicus) are

not active on GlcNAc [3–5] Tk-Dac de-N-acetylates

GlcNAc monomers as well as N-acetyl-chitooligomers

[7], but does not de-N-acetylate GlcNAc-6P or ManNAc

In contrast to other LmbE protein family members,

BC1534 and BC3461 are active on GlcNAc, GlcNAc2,

GlcNAc3, GlcNAc4, GlcNAc-1P, GlcNAc-6P, GalNAc,

ManNAc and UDP-GlcNAc, thus exhibiting a broader

substrate specificity compared to other LmbE protein

family members

The exact biological role of BC1534 and BC3461

proteins remains unclear Microarray data [29] and

deep sequencing [23] of the transcriptome showed that

the homologs in B anthracis (BA1557 and BA3524 for

BC1534 and BC3461, respectively) are expressed in

dif-ferent phases of the cell cycle (early to mid log phase

for BC1534 and late sporulation to early log phase for

BC3461) Of GlcNAc, GlcNAc2 and GlcNAc-6P,

which were tested for their docking properties in the

BC1534 active site, GlcNAc2had the lowest calculated

binding energy (data not shown), which is in

agree-ment with the kinetic parameters shown in Table 2

indicating that GlcNAc2 is the favored substrate RT-PCR experiments (data not shown) revealed similar expression profiles for BC1534 and BC3461 to that for an exochitinase (BC3725, EC 3.2.1.14) from

B cereus ATCC14579 This observation, in combina-tion with the reported chitinolytic activity of B cereus [30] and the presence of an endochitinase and a chito-sanase genes (bc0429 and bc2682 respectively) in its genome, support possible involvement of these enzymes in a chitinolytic pathway, similar to Tk-Dac

An alignment-based tree (Fig 6) placed both enzymes

in functional categories different from other LmbE proteins Analysis of the chromosome organization of bc1534 revealed the existence of a glycosyltransferase gene (bc1535) immediately downstream in the operon (Fig 4) This gene organization is common for most Firmicutes genomes, suggesting that the two proteins are functionally related in these organisms Interest-ingly, we observed that BC1534 is also active on UDP-GlcNAc As glycosyltransferases and UDP-GlcNAc are situated at a biosynthetic branch point leading to peptidoglycan formation, a possible role of BC1534 (and BC1535, EC 2.4.1.-) in modulating pepti-doglycan biosynthesis can be envisaged

In order to elucidate the importance of selected resi-dues in the catalytic mechanism, substrate affinity and specificity, as well as to investigate the importance of the oligomerization state on the activity of the enzyme, six point mutations and one fragment deletion were performed Central features of the catalytic mechanism are an H⁄ D pair (H110 ⁄ D112, Fig 3E) [10] that is proposed to play the role of a charge relay, and a hydrophilic pocket proposed as an ‘oxyanion hole’ (Y194, N150 and D108, Fig 3E) We tested the valid-ity of this hypothesis by mutating D112 to N and Y194 to F The variant D112N shown complete aboli-tion of catalytic efficiency against GlcNAc, and the

kcat against GlcNAc2 was decreased approximately 16 times with a subsequent decrease in Keff, indicating that the hydroxyl group of D112 plays a significant role in catalysis The Y194F variant exhibited broad substrate specificity similar to the native enzyme (Table 2) However in contrast to the native enzyme, it showed a dramatic increase (> 103) in Kmfor GlcNAc and GlcNAc2, and similar catalytic efficiency for both substrates These results suggest that the tyrosine hydroxyl group is directly associated with the enzyme’s affinity for the substrate

In order to test the suggestion that the loops sur-rounding the active site and the rim of the tunnel are directly implicated in determining the accessibility of the active site and the enzyme’s substrate specificity [10,20], we mutated three residues located on the rim

(R140, K207, A42) (Fig 3B–D,F) Automated docking

of GlcNAc2 substrate in the R140A active site showed

that the substrate was arranged in a loose way with

fewer hydrophobic interactions and a weaker hydrogen

bond compared with the native enzyme This different

orientation of the reaction site of the substrate could

explain the lower reported Keff values (Table 2) for

R140A In the native enzyme, the rim of the active site

tunnel is dominated by positively charged residues, i.e

R109, K154, K187, K207, K220 etc., that may drive

acetate out of the active site after the end of the

reac-tion The positioning of a negatively charged residue

(E140) could hamper the release of the acetate from

the active site This unfavorable behavior could

account for the lower catalytic reaction rates (kcat) in

the case of R140E (Table 2)

Surprisingly, the mutation K207I resulted in an

inac-tive enzyme K207 is located on the rim of the acinac-tive

site and it is quite unlikely that it has any influence on

the overall structure of the enzyme as it was found at

the edge of an a-helix (Fig 3F) K207 is not conserved

among other LmbE family members, indicating that

this residue is a unique feature of BC1534 that could

be related to the specificity of the enzyme

A42S exhibited lower Keff values, which could be

due to changes in interactions stabilizing the substrate

and⁄ or interactions with residues involved in the

cata-lytic mechanism (R53 and⁄ or D76) [10] (Fig 3D)

Deletion of the short b8-strand resulted in an insoluble

and inactive enzyme, supporting a previous suggestion

[10] that the structural building block of the BC1534 is a

dimer formed via b-strand exchange (Fig 3A)

Glucosamine (GlcN) is of importance in biomedicine

as it is used as dietary supplement for osteoarthritis

[31,32] Currently GlcN is produced by acid hydrolysis

of chitin extracted from crab and shrimp shells [33] A

new fermentation process utilizing E coli cells modified

by metabolic engineering for the production of

high-quality and low-cost GlcN has recently been reported

[34] The BC1534 R140A enzyme variant is potentially

a candidate for the enzymatic production of GlcN due

to its significantly increased kcattowards GlcNAc

In conclusion, we have biochemically characterized

two LmbE proteins from B cereus that exhibit the

broadest substrate specificity compared to other LmbE

protein family members, so far reported BC1534 and

BC3461 appear to have distinct functional roles, as

shown by their different expression profiles,

chromo-somal organization and sequence alignments Due to

their high similarity to their B anthracis homologs,

clarification of their biological roles will contribute to

a better understanding of the properties of this

life-threatening bacterium

Experimental procedures Materials

Primers were synthesized by the Microchemistry Facility of the Institute of Molecular Biology and Biotechnology (Heraklion, Greece) The expression plasmid pET26b and

E coli BL21 DE3 were obtained from Novagen (Merck, KGaA, Darmstadt, Germany) The expression plasmid pRSETA was purchased from Invitrogen (Carlsbad, CA, USA) E coli BL21 T7 Express lysY was purchased from New England Biolabs GmbH (Frankfurt, Germany) All chromatographic materials were obtained from GE Health-care Bio-Sciences AB (Uppsala, Sweden) Ni-nitrilotriacetic acid agarose, PCR and gel extraction kits were purchased from Qiagen (Valencia, CA, USA) Plasmid purification and RNA isolation kits were purchased from Macherey-Nagel GmbH & Co KG (Duren, Germany) The RT-PCR kit was obtained from Finnzymes Oy (Espoo, Finland) Substrates (including glycol chitosan) and common biochemicals were purchased from Sigma-Aldrich Ltd (St Louis, MO, USA) Restriction enzymes and DNA-modifying enzymes were purchased from MINOTECH Biotechnology (Heraklion, Greece) and New England Biolabs GmbH The instruments Fluostar Galaxy and Mastercycler Gradient (for PCR and RT-PCR) were purchased from BMG Labtechnologies GmbH (Offenburg, Germany) and Eppendorf Netheler-Hinz GmbH (Hamburg, Germany), respectively

Construction of expression plasmids bc1534 and bc3461 genes were isolated from B cereus ATCC14579 genomic DNA The primers used for bc1534 were BC1534-For (5¢-GGAATTCCATATGATGAGTGG-ATTACATATATTA-3¢; NdeI restriction site underlined) and BC1534-Rev (5¢-CCGCTCGAGTTTACATCCCCCT-AATAAATC-3¢; XhoI restriction site underlined) Plasmid pET26b was digested with NdeI and XhoI, and bc1534 was ligated into the corresponding sites, resulting in plasmid pET26b-bc1534 This plasmid construction was used for the production of BC1534 protein with a histidine tag at its C-terminus The primers used for bc3461 were BC3461-For (5¢-ATGGAGAGACATGTACTTGTT-3¢) and BC3461-Rev (5¢-CCGCTCGAGCTACTCCCATTTATAAGTCCA-3¢; XhoI restriction site underlined) Initial digestion of plasmid pRSETA with NdeI was followed by incubation with the Klenow fragment of DNA polymerase I, and finally diges-tion with XhoI The bc3461 gene was ligated into the corre-sponding sites of the plasmid

Production and purification of BC4361, BC1534 and BC1534 variants

To over-express bc1534, the plasmid pET26b-bc1534 was used for transformation of E coli BL21 DE3 The resultant