Mayer, Department of Biology, University of Konstanz, 78457 Konstanz, Germany Fax: +49 7531 88 3356 Tel: +49 7531 88 4854 E-mail: ch.mayer@uni-konstanz.de *Present address Department of
Trang 1b-N-acetylglucosaminidase/b-glucosidase from
Cellulomonas fimi
Christoph Mayer1,2,3, David J Vocadlo1,*, Melanie Mah2, Karen Rupitz1, Dominik Stoll2,
R A J Warren2 and Stephen G Withers1
1 Department of Chemistry, University of British Columbia, Vancouver, Canada
2 Department of Microbiology & Immunology, University of British Columbia, Vancouver, Canada
3 Department of Biology, University of Konstanz, Germany
Most enzymes catalyzing the hydrolysis of terminal
b-N-acetylglucosaminide linkages belong to families 3
and 20 of the glycoside hydrolases ([1,2] and the
glyco-side hydrolases database at URL http://afmb.cnrs-mrs
fr/CAZY/) Members of the two families greatly differ
in structure, enzyme mechanism, substrate specificity,
and physiologic function (for a review see [3] and
references cited therein) The enzymes in family 20 are designated as N-acetylhexosaminidases (EC 3.2.1.52) because they hydrolyze b-N-acetylgalactosaminides and b-N-acetylglucosaminides, with about a four-fold greater activity on the latter ([1], and references cited therein) b-N-Acetylglucosaminidases (EC 3.2.1.52) in family 3 are much more specific for the gluco-configuration,
Keywords
bifunctional glycosidase; cell wall recycling;
chitin metabolism; murein; peptidoglycan
Correspondence
C Mayer, Department of Biology, University
of Konstanz, 78457 Konstanz, Germany
Fax: +49 7531 88 3356
Tel: +49 7531 88 4854
E-mail: ch.mayer@uni-konstanz.de
*Present address
Department of Molecular Biology and
Biochemistry, Simon Fraser University,
Burnaby, BC, Canada
Database
The nucleotide sequences listed in this
paper have been submitted to the
DDBJ ⁄ EMBL ⁄ GenBank database under the
accession numbers AF478459 and
AF478460
(Received 22 February 2006, revised 3 May
2006, accepted 4 May 2006)
doi:10.1111/j.1742-4658.2006.05308.x
The Gram-positive soil bacterium Cellulomonas fimi is shown to produce at least two intracellular b-N-acetylglucosaminidases, a family 20 b-N-acetyl-hexosaminidase (Hex20), and a novel family 3-b-N-acetylglucosamini-dase⁄ b-glucosidase (Nag3), through screening of a genomic expression library, cloning of genes and analysis of their sequences Nag3 exhibits broad substrate specificity for substituents at the C2 position of the gly-cone: kcat⁄ Km values at 25C were 0.066 s)1Æmm)1 and 0.076 s)1Æmm)1 for 4¢-nitrophenyl b-N-acetyl-d-glucosaminide and 4¢-nitrophenyl b-d-glu-coside, respectively The first glycosidase with this broad specificity to be described, Nag3, suggests an interesting evolutionary link between b-N-ace-tylglucosaminidases and b-glucosidases of family 3 Reaction by a double-displacement mechanism was confirmed for Nag3 through the identification
of a glycosyl–enzyme species trapped with the slow substrate 2¢,4¢-dinitro-phenyl 2-deoxy-2-fluoro-b-d-glucopyranoside Hex20 requires the
acetami-do group at C2 of the substrate, being unable to cleave b-glucosides, since its mechanism involves an oxazolinium ion intermediate However, it is broad in its specificity for the d-glucosyl⁄ d-galactosyl configuration of the glycone: Km and kcat values were 53 lm and 482.3 s)1 for 4¢-nitrophenyl b-N-acetyl-d-glucosaminide and 66 lm and 129.1 s)1 for 4¢-nitrophenyl b-N-acetyl-d-galactosaminide
Abbreviations
DNP-2FGlc, 2¢,4¢-dinitrophenyl 2-deoxy-2-fluoro-b- D -glucopyranoside; Dp, degree of polarization; IPTG, isopropyl thiogalactopyranoside; 4MU-GlcNAc, 4¢-methylumbelliferyl b-N-acetyl- D -glucosaminide; pNP, 4-nitrophenol; pNP-Glc, 4¢-nitrophenyl b- D -glucopyranoside; pNP-GlcNAc, 4¢-nitrophenyl b-N-acetyl- D -glucosaminide; pNP-GalNAc, 4¢-nitrophenyl b-N-acetyl- D -galactosaminide; PVDF, polyvininylidene difluoride.
Trang 2exhibiting little if any activity on galactosyl substrates
[1,4–6] Family 3 primarily comprises b-glucosidases
(EC 3.2.1.21) and exo-b-glucanases (EC 3.2.1.58 and
3.2.1.74) However, b-N-acetylglucosaminidases form a
subgroup within family 3, characterized by the
sequence pattern K-H-(FI)-P-G-(HL)-G-x(4)-D-(ST)-H,
which is believed to be involved in binding of the
N-acetyl group [1,7]
The b-N-acetylglucosaminidases and hexosaminidases
in families 3 and 20 are both retaining enzymes, yet
they have different mechanisms [8] The family 20
enzymes do not form covalent glycosyl–enzyme
inter-mediates because they lack a nucleophilic carboxylate;
hydrolysis involves the anchimeric assistance of the
acetamido group of the substrate [8–11] By contrast,
family 3 enzymes do contain a nucleophilic carboxylate
and catalyze hydrolysis by a double-displacement
mechanism via a covalent glycosyl–enzyme
intermedi-ate [7,13–15] This mechanism is found in most
retain-ing glycosidases, e.g in lysozyme, an enzyme that
catalyzes an endo-type cleavage of the
N-acetylglucosa-mine-containing bacterial cell wall peptidoglycan [12]
The mechanism of family 3 exoglucanase ExoI from
Hordeum vulgareis understood in some detail [16] The
enzyme consists of two modules, one an (a⁄ b)8-barrel,
and the second a six-stranded b-sandwich [17,18] The
substrate binds to a pocket formed between the two
modules, with Asp285 of the first domain being the
catalytic nucleophile and Glu491 of the second domain
the acid–base catalyst, which accelerates the departure
of the aglycon by protonation of the glycosidic oxygen
[19] The catalytic nucleophile of a family 3
b-N-acetyl-glucosaminidase (ExoII) from Vibrio furnissii was
identified using the slow substrate
N-acetyl-5-fluoro-a-l-idopyranosaminyl fluoride [7] This residue is
con-served throughout family 3 An amino acid acting as
an acid–base catalyst in this enzyme is apparently
missing, since ExoII and other family 3
b-N-acetyl-glucosaminidases of Gram-negative bacteria comprise
only a single (a⁄ b)8-barrel module Generally, they
have molecular masses of about 35 kDa and are
pre-dicted to be cytoplasmic: the
b-N-acetylglucosamini-dase of Escherichia coli (NagZ) is a cytoplasmic
enzyme involved in peptidoglycan recycling [20,21]
Similar enzymes in other Gram-negative bacteria may
have the same function To date, only one family 3
b-N-acetylglucosaminidase-encoding gene (nagA) has
been cloned from a Gram-positive bacterium, namely
Streptomyces thermoviolaceus [6] This enzyme, like
most putative family 3 b-N-acetylglucosaminidases of
Gram-positive bacteria, has a molecular mass of about
60 kDa and comprises two modules It is extracellular
and thought to be involved in chitin degradation
Chitin is degraded by the concerted action of chi-tinase(s) (EC 3.2.1.14) and b-N-acetylhexosamini-dase(s), which may involve other proteins [22–27]
As part of an analysis of the mechanisms and func-tions of N-acetylglucosaminidases of Gram-positive bacteria, this article reports the cloning and sequencing
of two genes from the Gram-positive soil bacterium Cellulomonas fimi that encode enzymes acting on ter-minal b-N-acetylglucosamine residues: a family 20 b-N-acetylhexosaminidase (Hex20) and a novel family
3 b-N-acetylglucosaminidase⁄ b-glucosidase (Nag3) Nag3 is the first b-glycosylase to be described that lacks specificity for substituents at C-2
Results
Detection of b-N-acetylglucosaminidase activity
in Cellulomonas fimi cell extracts Cellulomonas fimi grows on minimal medium supple-mented with 0.2% (w⁄ v) chitin as the sole source of carbon and it secretes a chitinase (C Mayer, unpub-lished results) However, b-N-acetylglucosaminidase activity assayed with chromogenic substrates could only be detected in the soluble cell fraction; a specific activity of 0.20 ± 0.05 UnitsÆmg)1 with 4¢-methylum-belliferyl b-N-acetyl-d-glucosaminide (4MU-GlcNAc) was determined within the soluble cell extract The intracellular b-N-acetylglucosaminidase(s) of Cellulo-monas fimi could not be induced by addition of chitin
or chitosan (0.2% w⁄ v) to the growth medium How-ever, significantly higher b-N-acetylglucosaminidase activity (0.34 ± 0.05 UÆmg)1) was measured when 0.05% (w⁄ v) N-acetylglucosamine was added to the growth medium Glucose in the culture medium had
no catabolic repression effect To identify and clone the gene(s) encoding for intracellular b-N-acetylglu-cosaminidase(s), a Cellulomonas fimi genomic expres-sion library was screened
Screening of a Cellulomonas fimi genomic library
A Cellulomonas fimi genomic library was prepared pre-viously by inserting genomic DNA fragments (2–
5 kbp) into the EcoRI site of the multiple cloning site
of lambda ZAPII (Stratagene [28,29]) This created fusions of the genomic inserts with the first 36 amino acids of the E coli b-galactosidase coding sequence transcribed from the lacZ promoter E coli XLOLR cells transformed with the excised phagemid library were screened for isopropyl thiogalactopyranoside (IPTG)-inducible expression of b-N-acetylglucosamini-dase activity using 4MU-GlcNAc Five positive clones
Trang 3were isolated from two independent screenings Three
clones (designated CF2, 3 and 10) produced intensely
fluorescent halos, whereas the other two colonies (CF5
and 13) produced weakly fluorescent haloes
Restric-tion endonuclease digesRestric-tion showed that the plasmids
in the clones carried inserts of the following sizes:
2.8 kb (pCF5), 2.2 kb (pCF2 and pCF3), 2.0 kb
(pCF10), and 1.7 kb (pCF13) By restriction mapping,
pCF2 and pCF3 were found to contain an identical
2.2 kb insert, which contained the 2.0 kb insert of
pCF10 DNA sequencing of the inserts revealed the
2.0 kb insert to be an incomplete ORF missing 20 bp
at the 5¢ end and a 200 bp portion at the 3¢ end
Plas-mid pCF5 carried a 2.8 kb insert containing the
com-plete insert (1.7 kb) in pCF13
Sequence alignment and classification to family
20 glycoside hydrolases
The 2.2 kb Cellulomonas fimi genomic DNA fragment
of pCF2 carries a 1491 kb ORF with a G⁄ C content
of 73.3% that starts with a GTG codon and ends with
a TGA stop codon A putative ribosome-binding site
(Shine–Dalgarno sequence) was found six bases
upstream of the start codon The deduced amino acid
sequence of the encoded protein, designated Hex20,
had high similarity (38% overall sequence identity
according to the blast sequence alignment tool) to a
b-N-acetylhexosaminidase from Streptomyces plicatus
(UniProt database identifier O85361) as well as other
family 20 glycoside hydrolases Recently, the crystal
structure of Streptomyces plicatus
b-N-acetylhexosa-minidase was determined ([9]; structure identifier
1HP4): the catalytic C-terminal module forms a
(b⁄ a)8-barrel-type (TIM-barrel) structure, first
elucida-ted for the Serratia marcescens chitobiase [30], and the
N-terminal module forms a a + b sandwich structure
A multiple sequence alignment of the
b-N-acetylhexos-aminidases from Cellulomonas fimi, Streptomyces
plica-tusand Streptomyces thermoviolaceus (NagB, Q9RHV6
[31]), as well as a highly similar putative enzyme from
Streptomyces coelicolor (Q9L068), along with the
sec-ondary structural elements of 1HP4, are given in
Fig 1 Regions within Hex20 that differ strongly from
comparable regions within the Streptomyces plicatus
enzyme are found in the N-terminal module of
unknown function and within the following regions of
the catalytic module: a-helix 4 and the loops after
b-strands 4 and 6 These parts of the catalytic (ba)8
-barrel are believed to constitute the aglycon-binding
site of the enzymes However, we do not know if these
differences in sequence lead to distinct aglycon
specifi-cities of the enzymes
Sequence alignment and classification to family 3 glycoside hydrolases
The 2.8 kb Cellulomonas fimi genomic DNA fragment
of pCF4 (¼ pCF13) contained a 1695 bp open reading frame (ORF) with a G⁄ C content of 70.3%, starting with an ATG codon and ending with a TGA stop codon A putative ribosome-binding site (Shine–Dalg-arno sequence) was found upstream of the start codon The deduced amino acid sequence of the protein, enco-ded by the 1695 bp ORF, designated Nag3, had some 25% overall sequence identity to b-N-acetylglucosa-minidase NagA from Steptomyces thermoviolaceus (O82840) and similarity to other members of the b-N-acetylglucosaminidase subfamily of family 3 glyco-side hydrolases (Figs 2,3) Nag3 may be part of an operon; there are putative ORFs upstream and down-stream of the 1695 bp ORF The updown-stream ORF showed similarities to ABC transport proteins and the downstream ORF showed similarities to haloacid deh-alogenase-like hydrolases (HAD superfamily) The stop codon (TGA) of the putative upstream ORF overlaps the start codon of the 1695 bp ORF
Subcloning, overexpression and N-terminal protein sequencing
The genes hex20 and nag3 were subcloned into the expression vector pET29b, which allowed heterologous overexpression of the Cellulomonas fimi enzymes in
E coli BL21(DE3) cells Typically, about 100 mg of pure His6-tag fusion proteins (Hex20 and Nag3) were obtainable from 1 L of LB culture Overexpression of Nag3 was enhanced by growth of E coli cells at reduced temperature (25C) after induction with IPTG The N-terminal amino acid sequences of the purified proteins were identical to those deduced from the nuc-leotide sequences (italics in Figs 1 and 2) It should be noted that the GTG start codon obtained for hex20 was exchanged with ATG for expression in E coli (Fig 1)
Characterization of the purified enzymes Purified Hex20 and Nag3 His6-fusion proteins were active on 4MU-GlcNAc, which is the fluorogenic sub-strate used for the screening In addition, they released 4-nitrophenol (pNP) from the chromogenic substrate 4¢-nitrophenyl b-N-acetyl-d-glucosaminide (pNP-Glc-NAc) The huge differences in activity already observed throughout the screening were confirmed with purified protein The kinetic parameters of Hex20 and Nag3 for pNP-glycosides are presented in Table 1 Hex20 was highly active on both b-N-acetylglucosaminide
Trang 4and b-N-acetylgalactosaminide (Fig 4A): Km and kcat
values were 53 lm and 482.3 s)1 for pNP-GlcNAc,
and 66 lm and 129.1 s)1 for p-nitrophenyl
b-N-acetyl-galactosaminide (pNP-GalNAc) at 25C An activity
ratio (pNP-GlcNAc⁄ pNP-GalNAc) of 3.7 was
deter-mined, a value in the range commonly observed for
hydrolysis of these substrates by family 20
b-N-acetyl-hexosaminidases [1]
A high Km value and a low kcat value were
deter-mined for Nag3 with pNP-GlcNAc: the Km value was
2.7 mm and the kcat value at 25C was 0.18 s)1 These values are in the range observed for other fam-ily 3 N-acetylglucosaminidases [4,6], which generally have very low specific activity Nag3 was also found
to be active on 4¢-nitrophenyl b-d-glucopyranoside (pNP-Glc) (Fig 4B) However, there was a linear rela-tionship of enzyme velocity with pNP-Glc concen-tration up to 24 mm, the limit of solubility of the substrate, so the Kmand kcatvalues could not be deter-mined for pNP-Glc Interestingly, the values reflecting
Fig 2 Multiple amino acid sequence alignment of Nag3 of Cellulomonas fimi (Q7WUL4_CELFI) and selected family 3 b-N-acetylglucosami-nidases: NagA of Streptomyces thermoviolaceus (Q82840_STRTH) and HexA from Alteromonas sp (P48823_ALTSO) and the sequences of three putative b-N-acetylglucosaminidases from Bacillus subtilis (P40406_BACSU), Streptomyces colicolor (Q9RDG9_STRCO) and Clostridium perfringens (HEXA_CLOPE) The conserved catalytic nucleophile residue (r) identified in ExoII from Vibrio furnissii (31) and the sequence identifier (16) of the N-acetylglucosaminidase subgroup of family 3 glycoside hydrolases (*, bold letters) are indicated For definitions see also legend to Fig 1.
Fig 1 Multiple amino acid sequence align-ment of Hex20 of Cellulomonas fimi (Q7WUL4_CELFI) and selected family 20 b-N-acetylhexosaminidases: NagB of Strep-tomyces thermoviolaceus (Q9RHV6_STRTL), Hex of Streptomyces plicatus (O85361_ STRPL), and a putative b-N-acetylhexos-aminidase of Streptomyces coelicolor (Q9L068_STRCO) The abbreviations used reference the accession numbers of the UniProt database and the organism codes Dark shading indicates highly conserved res-idues, and light shading indicates conserved similar residues Alignment was generated using CLUSTALW [46], and shading was per-formed with version 3.21 of BOXSHADE (by
K Hofmann and M Baron) The N-terminal amino acid sequence of Hex20 from Cellulo-monas fimi that was confirmed by sequen-cing is shown in italics; the GTG start codon obtained for the native hex20 was
exchanged with ATG for expression in Escherichia coli Underlined are the (puta-tive) cleavage sites of the signal sequences Secondary structural elements of the Strep-tomyces plicatus enzyme [9] are indicated: b-sheet (¼), a-helix (//) and the structural elements of the N-terminal catalytic (ab)8 -barrel The conserved catalytic acid ⁄ base residue (r) and the cysteine residues form-ing an intramolecular disulfide bridge in the b-N-acetylglucosaminidase of Streptomyces plicatus (*) are indicated.
Trang 6the catalytic efficiency (kcat⁄ Km) determined for
pNP-GlcNAc and pNP-Glc were about the same for Nag3
(Table 1)
Hex20 hydrolyzed N-acetylchitooligomers (degree of
polarization (Dp) 2–6) at about the same rate, as
ana-lyzed by TLC (supplementary Fig S1) However,
Nag3 did not release GlcNAc from chitobiose⁄
N-ace-tylchitooligomers and Glc from cellobiose⁄
b-glucan-ol-igomers (data not shown)
Stability and pH effect Hex20 was stable at pH 6.0–9.5, retaining its activity for several months when stored in the elution buffer used for nickel chelate chromatography (20 mm sodium phosphate⁄ 80 mm imidazole pH 7.5 and
300 mm NaCl) at 4C However, the enzyme was rapidly inactivated above pH 9.5 Nag3 precipitated below pH 6.0; it was resonably stable between pH 6.8
0.1
Q9XEI3/EXOI HORVU P33363/BGLX ECOLI
Q9P8F4/BGLA ASPNG
P96157/EXOII VIBFU
P75949/NAGZ ECOLI
P48823/HEXA ALTSO
P40406/YBBD BACSU
082840/NAGA STRTL Q9RDG9 STRCO
Q8XP12 CLOPE Q7WUL3/NAG3 CELFI
Q8W012/ARAI HORVU Q8W011/XYLA HORVU Q42835/EXOII HORVU
Fig 3 Cladogram showing the evolutionary relationship of Nag3 of Cellulomonas fimi (Q7WUL3 ⁄ NAG3_CELFI) and selected members of family 3 of glycoside hydrolases The abbreviations used reference the accession numbers of the UniProt database and the organism codes: NagA of Streptomyces thermoviolaceus (Q82840 ⁄ NAGA_STRTH) and HexA from Alteromonas sp (P48823 ⁄ HEXA_ALTSO) and the sequences of three putative b-N-acetylglucosaminidases from Bacillus subtilis (P40406 ⁄ YBBD_BACSU), Streptomyces colicolor (Q9RDG9_STRCO) and Clostridium perfringens (HEXA_CLOPE) (see Fig 3); the b-N-acetylglucosaminidases of two Gram-negative bacteria, NagZ of Escherichia coli (P75949 ⁄ NAGZ_ECOLI) and ExoII of Vibrio furnisii (P96157 ⁄ EXOII_VIBFU); members of the b-glucosidase subfamily, b-glucosidase X of Escherichia coli (P33363 ⁄ BGLX_ECOLI) and b-glucosidase A of Aspergillus niger (Q9P8F4 ⁄ BGLA_ASPNG), the two exoglucanases ExoI and ExoII of Hordeum vulgare (Q9XEI3 ⁄ EXOI_HORVU and Q42835 ⁄ EXOII_HORVU), and a b-xylosidase and an a- L -arabi-ofuranosidase ⁄ b-xylosidase of Hordeum vulgare (Q8W011 ⁄ XYLA_HORVU and (QW012 ⁄ ARAI_HORVU).Nag3 and the putative family 3 N-acetylglucosaminidase of Clostridium perfringens (Q8XP12) form an intermediate branch between b-glucosidases and b-N-acetylglucosami-nidases of family 3 The phylogenetic tree was created with the program TREEVIEW (by R D M Page).
Table 1 Kinetic parameters for the reactions of Cellulomonas fimi b-N-acetylhexosaminidase (Hex20) and b-N-acetylglucosaminidases (Nag3) with pNP glycosides The enzymic reaction was carried out in 50 m M sodium phosphate buffer (pH 7.08) at 25 C The molar extinction coef-ficient ( M )1Æcm)1) at 400 nm for pNP was 7280 Standard errors for the values of K
m and kcatmeasured here were less than 5%, except where standard error values are indicated.aNot determined due to a reaction being too slow to be detected.bNot determined due to the linear relationship of enzyme velocity with substrate concentration pNP-GlcNAc, 4¢-nitrophenyl b-N-acetyl- D -glucosaminide; pNP-GalNAc, 4¢-nitrophenyl b-N-acetyl- D -galactosaminide; pNP-Glc, 4¢-nitrophenyl b- D -glucopyranoside.
Substrate
Km(l M ) kcat(s)1) kcat⁄ K m (s)1Æl M )1) K
m (l M ) kcat(s)1) kcat⁄ K m (s)1Æl M )1)
Trang 7and 8.4; however, it lost its catalytic activity in diluted
buffers within a day at 4C The half-life of Nag3 at
room temperature was only a few hours Addition of
sodium chloride (0.5 m), dithiothreitol (0.1 and 1 mm),
BSA (0.5 mgÆmL)1), sucrose and trehalose (20%) had
no huge effect on Hex3 stability (Table 2) However, adding glycerol and⁄ or phosphate stabilized the enzymes, and Nag3 retained its activity for several
0.00
0.05
0.10
A 400
0.15
0.20
0.00 0.25 0.50 0.75 1.00 1.25
-20 0 20 40 60 80 100 120 140 160 25
50 75 100
A
0.05 0.10 0.15 0.20 0.25 0.30 0.35
50 100 150
B
Fig 4 Michaelis–Menten plot of initial rates of hydrolysis of (A) 4¢-nitrophenyl b-N-acetyl- D -glucosaminide (pNP-GlcNAc) (d) and 4¢-nitrophe-nyl b-N-acetyl- D -galactosaminide (pNP-GalNAc) (s) by Cellulomonas fimi Hex20 (5.59 · 10)5mg ⁄ mL) at 25.2 C and pH 7.08 and (B)
pNP-GlcNAc (n) and pNP-Glc (h) by Cellulomonas fimi Nag3 (3.09· 10)3mg ⁄ mL) at 25.2 C and pH 7.08 Inset: graphical analysis of K m and kcat
by Lineweaver–Burk linearization.
Table 2 Effects of various reagents on the stability of Cellulomonas fimi Nag3 dithiothreitol.
% Remaining relative activity a (18 h incubation) b
Remaining relative activity a (90 h incubation) b
60 m M imidazol pH 7.5
a
The enzymic reaction was carried out in 20 m M Tris ⁄ HCl buffer (pH 7.3) at 25 C with 4¢-nitrophenyl b-N-acetyl- D -glucosaminide (pNP-Glc-NAc) (6.5 m M ) b Before assaying, Nag3 was incubated for 18 and 90 h with the indicated supplement; c the activity measured after incubation for the indicated time with the supplement shown in bold was set at 100%.
Trang 8months when stored in glycerol (20% final
concentra-tion) and phosphate buffer at pH 7.3 and) 20 C
Ex-oII, a family 3 N-acetylglucosaminidase from Vibrio
furnissi, is activated by 400–700 mm sodium chloride
[4] However, sodium chloride up to 700 mm had no
effect on Hex3; there was a 10% decrease in activity
with 1 m NaCl
The pH dependence of Hex20 and Nag3 was
investi-gated using pNP-GlcNAc and pNP-Glc, respectively,
over a pH range of 6.2–9.2 and 6.8–8.5, respectively
Hex20 showed a broad, bell-shaped pH optimum curve
with a maximum between pH 7.3 and 8.7 with
pNP-GlcNAc and half-maximal rate at about pH 7 and 9
(Fig 5A) By contrast, the family 20
b-N-acetylhexosa-minidase from Streptomyces plicatus has a pH
optimum of 5 on pNP-GlcNAc [11] The kcat⁄ Km for
Hex20 was dependent on two ionizable groups with
pKavalues of 6.9 and 8.8 (Fig 6B) Nag3 gave a com-plex pH profile on pNP-Glc, with a narrow maximal rate at pH 7.3 and half-maximal rates at about pH 6.8 and 8.0 (Fig 5) This is consistent with the pH opti-mum determined for the b-N-acetylglucosaminidase (ExoII) from Vibrio furnissii [4] The pKa of the lower ionization constant was 6.7; however, a value for an upper ionization could not be determined from the data (Fig 5B)
MS and labeling The mass of purified Hex20 was 54 186 Da, as ana-lyzed by ESI⁄ MS, which is in perfect agreement with
0.00
0.02
0.04
0 2500 5000 7500
A
kca
–1 m
Kcat
-1
0
1
2
3
-4 -3 -2 -1
B
pH
Fig 5 pH dependence of k cat ⁄ K m for the Nag3- and
Hex20-cata-lyzed reaction (A) The pH profiles of Nag3 (d, left scale) and
Hex20 (s, right scale) were determined using pNP-Glc and
4¢-nitro-phenyl b-N-acetyl- D -glucosaminide (pNP-GlcNAc), respectively, at
25 C The reaction buffers were 100 m M sodium citrate⁄
phos-phate (pH 6.0–7.3), 100 m M sodium phosphate (pH 7.0–8.2) and
100 m M glycine ⁄ HCl (pH 7.8–10) (B) Shows the same data used to
fit Eqn (1); the lines represent the best fit of the equation to the
pkcat⁄ K m data (Nag3, pKa1¼ 6.70 ± 0.33; pK a2 could not be
deter-mined from the data; Hex20, pKa1¼ 6.91 ± 0.10; pKa2¼
8.79 ± 0.12).
B
C
60971.0
61126.0
61135.0
mass (Da)
62000
50
0 20
10
0 20
10
0
Fig 6 Transform of the electrospray mass spectrum of (A) Nag3, and (B) and (C) Nag3 incubated at room temperature with 10 m M
2¢,4¢-dinitrophenyl-2-deoxy-2-fluoro-b-glucose for 4 h and 20 h, respectively The mass shifts (157 and 166) of peaks shown in (B) and (C) compared to the peak shown in (A) correspond to a 2-de-oxy-2-fluoro-b-glucosyl residue (162 Da) covalently bound to Nag3.
Trang 9the theoretical mass of the cloned enzyme (54 186 Da).
The mass of the purified Nag3 protein was determined
by ESI⁄ MS to be 60 971 Da, close to the theoretical
mass of the cloned enzyme (60 945 Da) After
incuba-tion with 2¢,4¢-dinitrophenyl
2-deoxy-2-fluoro-b-d-glucopyranoside (DNP-2FGlc), two species are
observed: the native, unlabeled enzyme, and another
species with a mass of 61 126–61 135 Da (Fig 6) The
mass difference observed between the native and
inhib-ited enzyme is 164 Da, a value that is consistent,
within error, with the addition of a single
2-deoxy-2-fluoroglucosyl label (162 Da) The rate of the labeling
was consistent with the expectation of slow
inactiva-tion by the inhibitor when the low apparent kcatvalues
for Nag3 with chromogenic glucosides are kept in
mind Prolonged incubation of the enzyme with the
inhibitor leads to almost complete inactivation of the
native enzyme The observation of a covalent glycosyl
intermediate provides strong evidence for a mechanism
involving an enzymic nucleophile, as shown previously
for two family 3 glycoside hydrolases: the single
domain b-N-acetylglucosaminidase from V furnissii [7]
and the two domain b-glucosidases from Aspergillus
niger [13] Sequence alignment using the clustal w
algorithm revealed a conserved aspartate residue
within the sequence GLVVSDS to be the putative
cat-alytic nucleophile By contrast, the hydrolytic
mechan-ism of retaining family 20 b-N-acetylhexosaminidases
involves the assistance of the acetamido group of the
substrate [8,9,11]
Discussion
Cellulomonas fimi is strongly cellulolytic, producing a
complex cellulose degradative system The system,
comprising mostly extracellular enzymes, is understood
in considerable detail (e.g [29,32–38]) Cellulomonas
fimi also degrades chitin (C Mayer, unpublished
observation), a homopolymer of GlcNAc similar to
cellulose, but nothing is known of its chitinolytic
sys-tem Recently, a chitinase was isolated from culture
supernatant of Cellulomonas flavigena [39] and a
chi-tinase-encoding gene was cloned from Cellulomonas
uda [40] Cellulomonas fimi also secretes one (or more)
chitinase(s) (C Mayer, unpublished observation), but
N-acetylglucosaminidase activity is present only in the
soluble cell extract Of the two enzymes described
here, only Hex20 degrades N-acetylchitooligomers and
may be involved in chitin degradation The function
of Nag3 is unclear It has low catalytic activity relative
to Hex20 on chromogenic substrates: the catalytic
effi-ciency (kcat⁄ Km value) for hydrolysis of pNP-GlcNAc
by the two enzymes differs by a factor of 105
(Table 1) In this respect, Nag3 resembles family 3 N-acetylglucosaminidases of Gram-negative bacteria, which are involved in cell wall (peptidoglycan) recyc-ling [1,7,20,21,41] However, Nag3 is unusual in that it acts on b-N-acetyl-d-glucosaminides and b-d-gluco-sides, so it should be referred to as a b-N-acetyl-d-glu-cosaminidase⁄ b-d-glucosidase The catalytic efficiencies against pNP-Glc and pNP-GlcNAc were similar, seem-ingly a consequence of much higher apparent values for both kcat and Km for the b-glucoside Unfortu-nately, the exact kinetic parameters for hydrolysis of pNP-Glc by Nag3 could not be determined because the enzyme was not saturated with the substrate within the limits of its solubility Although a family 3 enzyme from barley was characterized that was referred to as
a ‘bifunctional’ a-l-arabinofuranosidase⁄ b-d-xylopyra-nosidase [42], there is, to our knowledge, no previous report on an enzyme with equivalent b-glucosidase and b-N-acetylglucosaminidase activity
Glycosyl hydrolases of family 3 form two distinct subgroups: a b-glucosidase subfamily and a b-N-acetyl-glucosaminidase subfamily (Fig 3) Being a ‘bifunc-tional’ b-N-acetyl-d-glucosaminidase⁄ b-d-glucosidase, Nag3 of Cellulomonas fimi represents an interesting link between the b-N-acetylglucosaminidase and the b-glucosidase branch of family 3 of glycoside
hydrolas-es A conserved sequence motif in the b-N-acetylglu-cosaminidase subgroup within family 3 may represent the N-acetyl group-binding site [1,7] Interest-ingly, Nag3, as well as the uncharacterized family 3 enzyme (Q8XP12) within the genome of the recently sequenced bacterium Clostridium perfringens [43], show
a significant alteration within this motif: the K-H-(FI)-P-G-(HL)-G-x(4)-D-(ST)-H motif is changed to K-H-(FI)-P-G-D-G-x(4)-D-Q-H (Fig 2) It can be spe-culated that changes within this motif (underlined) are responsible for the broad substrate specificity of Cellu-lomonas fimi Nag3 for substitution of the C2 position, and further studies in order to confirm this thesis are under way A hint for a possible function of Nag3 comes from a gene neighbor analysis of a putative b-N-acetylglucosaminidase of Clostridium perfringens using the European Molecular Biology Laboratory Search Tool for the Retrieval of Interacting Genes⁄ Proteins (string) Analysis revealed that the encoding gene is connected to a cluster of genes similar to known genes involved in the uptake and metabolism
of glucuronides We speculate that the putative N-ace-tylglucosaminidase of Clostridium perfringens and poss-ibly also Nag3 of Cellulomonas fimi might be involved
in the degradation of glucuronic acid-containing gly-cosaminoglycans such as hyaluronic acid Preliminary experiments, however, could not confirm this hypothesis;
Trang 10hydrolysis of hyaluronic acid by Nag3 could not be
detected by TLC analysis b-N-Acetylglucosaminidases
involved in hyaluronic acid degradation have been
placed into family 84 of glycoside hydrolases rather
than family 3 [44] N-Acetylglucosaminidases in family
84, like family 20 enzymes, use a catalytic mechanism
involving anchimeric assistance of the 2-acetamido
group of the substrate However,
N-acetylglucosami-nidases in family 3 are retaining enzymes that use a
double-displacement mechanism involving the
partici-pation of a catalytic nucleophilic group in the enzyme
active site [7,13] Our data confirm that Cellulomonas
fimi Nag3 acts by participation of a catalytic
nucleo-phile: incubation of Nag3 with DNP-2FGlc permits
the observation by ESI⁄ MS of a high steady-state
population of a 2-deoxy-2-fluoroglucosyl–enzyme
inter-mediate The identification of a bifunctional
b-N-ace-tyl-d-glucosaminidase⁄ b-d-glucosidase is an interesting
example of divergent evolution towards new substrate
specificity within family 3 of glycoside hydrolases
Fur-ther structural and mutational studies are required to
elucidate the basis of substrate specificity in this family
of glycoside hydrolases
Experimental procedures
Materials
Chemicals, reagents and materials were purchased as
fol-lows: growth media components from Difco (Sparks, ML);
DNA purification kits from Qiagen (Hilden, Germany);
restriction endonucleases, DNA ligase and DNA
poly-merase from New England Biolabs (Beverly, MA) and
Roche-Boehringer Mannheim (Germany); and His-bind
metal chelation resin from Novagen (Madison, WI)
Oligo-nucleotides were synthesized, and DNA and protein
sequences determined by the Nucleic Acids and Peptide
Service (NAPS) Unit of the Biotechnology Laboratory at
the University of British Columbia
N-Acetylchitooligosac-charides (Dp 2–6) were from Seikagaku America
hyaluronic acid were from Sigma 4MU-GlcNAc and
DNP-2FGlc were synthesized by standard procedures
Bacterial strains, plasmids and phages
E colistrain BL21(DE3) and pET29b were from Novagen
(Madison, WI) E coli XLOLR and the library (2–5 kbp
fragment length) of genomic DNA from Cellulomonas fimi
in k zapii (18, 19) were from Stratagene (La Jolla, CA)
Cultures were grown in LB medium supplemented with
50 mgÆL)1 ampicillin, or TYP medium (tryptone 16 gÆL)1,
yeast extract 16 gÆL)1, NaCl 5 gÆL)1, K2HPO4 2.5 gÆL)1)
containing 50 mgÆL)1kanamycin
Screening and isolation of Cellulomonas fimi genes encoding N-acetylglucosaminidases Plasmid isolations, restriction enzyme digests, ligations and transformations were performed using standard techniques Phagemids (pBluescript SK) were excised from the k ZAPII library using a helper phage and transferred to E coli XLOLR according to the supplier’s protocol Sufficient cells to yield about 500 colonies per plate were spread on
LB ampicillin agar After incubation for two days at 37C, colonies were replicated on LB ampicillin agar supplemen-ted with 4MU-GlcNAc (200 mgÆL)1) and isopropyl thiogal-actopyranoside (IPTG; 1 mm) It was necessary to screen replicas because the 4-methylumbelliferone product released
by enzyme action appeared to be toxic to the cells Colonies were screened for fluorescence at 366 nm using a UV trans-illuminator Nucleotide sequences of inserts were deter-mined by primer walking and confirmed by sequencing the complementary strand The nucleotide sequences of hex20A and nag3A have been submitted to the DDBJ⁄ EMBL ⁄ GenBank databases under the accession numbers AF478459 and AF478460 The UniProt database accession numbers are Q7WUL4 and Q7WUL3, repectively The GenBank and SWISS-PROT databases were used for nuc-leotide and amino acid sequence searches using the basic local alignment search tool (blast)
Construction of pETcfnag3 and pETcfhex20 The putative N-acetylglucosaminidase genes within the inserts in pCF2 and pCF5 were amplified by PCR using oligonucleotide primers based on the ORF sequences (under-lined are the restriction sites NdeI, NotI and XhoI introduced
by the primer): CF2NdeI 5¢-CC CAT ATG CCC GAC GTC GCC GTC ATC C-3¢; CF2NotI 5¢-TT GCG GCC
CAT ATG ATC GAC CTG ACC GCA GCC-3¢; CF5XhoI
PCR mixtures contained 10 lm primers, 1 mm each deoxyri-bonucleoside triphosphate, 50 ng of phagemid DNA, 5 U
of Pwo polymerase and 4% DMSO in 100 lL of DNA polymerase buffer Thirty PCR cycles (45 s at 94C, 45 s at
63C, and 120 s at 72 C) were performed in a thermal cycler (Perkin Elmer Applied Biosystems, Boston, MA, USA, GeneAmp PCR System 2400) The amplified frag-ments were cloned into pET29b according to a protocol des-cribed previously [45]
Production, purification and N-terminal sequencing of recombinant proteins
E coli BL21(DE3) carrying pET29cfnag3 or pET29cfhex20 was grown at 37C in TYP kanamycin to a D600 nm value
of 0.6–0.8, IPTG was added to a concentration of 1 mm, and incubation was continued for a further 12 h at 28C