Novel β-N-acetylglucosaminidases from Vibrio harveyi 650: Cloning, expression, enzymatic properties, and subsite identification
© Suginta et al; licensee BioMed Central Ltd. 2010
Received: 4 May 2010
Accepted: 29 September 2010
Published: 29 September 2010
Since chitin is a highly abundant natural biopolymer, many attempts have been made to convert this insoluble polysaccharide into commercially valuable products using chitinases and β-N-acetylglucosaminidases (GlcNAcases). We have previously reported the structure and function of chitinase A from Vibrio harveyi 650. This study t reports the identification of two GlcNAcases from the same organism and their detailed functional characterization.
The genes encoding two new members of family-20 GlcNAcases were isolated from the genome of V. harveyi 650, cloned and expressed at a high level in E. coli. Vh Nag1 has a molecular mass of 89 kDa and an optimum pH of 7.5, whereas Vh Nag2 has a molecular mass of 73 kDa and an optimum pH of 7.0. The recombinant GlcNAcases were found to hydrolyze all the natural substrates, Vh Nag2 being ten-fold more active than Vh Nag1. Product analysis by TLC and quantitative HPLC suggested that Vh Nag2 degraded chitooligosaccharides in a sequential manner, its highest activity being with chitotetraose. Kinetic modeling of the enzymic reaction revealed that binding at subsites (-2) and (+4) had unfavorable (positive) binding free energy changes and that the binding pocket of Vh Nag2 contains four GlcNAc binding subsites, designated (-1),(+1),(+2), and (+3).
Two novel GlcNAcases were identified as exolytic enzymes that degraded chitin oligosaccharides, releasing GlcNAc as the end product. In living cells, these intracellular enzymes may work after endolytic chitinases to complete chitin degradation. The availability of the two GlcNAcases, together with the previously-reported chitinase A from the same organism, suggests that a systematic development of the chitin-degrading enzymes may provide a valuable tool in commercial chitin bioconversion.
Chitin is a β-1,4-linked homopolymer of N-acetylglucosamine (GlcNAc), which is found mainly in the exoskeleton of crustaceans, insects and in the cell walls of fungi. Chitin is one of the most abundant polymers in nature and its degradation derivatives are pharmaceutically valuable. for example, chitoligosaccharides can stimulate the immune system to respond to microbial infections and chitin monomers have been shown to act as anti-aging and anti-tumor agents, as well as to relieve the symptoms of osteoarthritis [1–6]. Complete degradation of chitin requires chitinases (EC 220.127.116.11) and β-N-acetylglucosaminidases (GlcNAcases) or chitobiases (EC 18.104.22.168), so such enzymes could potentially serve as biocatalysts in the production of chitin derivatives of desired sizes during the recycling of chitin biomass.
As well as functioning in chitin degradation by bacteria, GlcNAcases are also known to be key enzymes in the catabolism of glycoconjugates containing N-acetylglucosamine residues [7, 8] and mutations of the gene encoding a human GlcNAcase homologue (HexA) cause a fatal genetic lipid storage disorder, known as Tay-Sachs disease . In the CAZy database (http://www.cazy.org), GlcNAcases are classified into glycosyl hydrolases family 3 (GH-3) or family 20 (GH-20), which differ in sequence and mode of enzyme action [10, 11]. Family-3 GlcNAcases are thought to act by a standard retaining mechanism involving a covalent glycosyl-enzyme intermediate while family-20 enzymes employ a 'substrate-assisted' mechanism involving the transient formation of an oxazolinium ion intermediate [12–15]. Most of the GlcNAcases described hitherto belong to the GH-20 family. To date, only five bacterial GH-3 GlcNAcases have been characterized, including NagZ or ExoII from Vibrio furnissii , Nag3A from Clostridium paraputrificum M-2 , NagA from Streptomyces thermoviolaceus , and NagA and CbsA from Thermotoga maritima and T. neapolitana .
Vibrio harveyi, formerly known as V. carchariae, is a Gram-negative marine bacterium that causes luminous Vibriosis, a serious disease that affects commercially farmed fish and shellfish species [20, 21]. We previously reported isolation of the gene encoding endochitinase A from Vibrio harveyi type strain 650 for functional and structural characterization [22, 23]. In this study, we employed a homology-based strategy to isolate two GlcNAcase genes from the genome of the same Vibrio strain. Sequence analysis suggested that the resultant polypeptides were new members of the GH-20 family. Enzymic properties of the GlcNAcases expressed in E. coli were investigated. Their kinetic properties and identification of the subsites in the more active enzyme are discussed in further detail.
Results and Discussion
Gene isolation and sequence analysis
Recombinant expression and mass identification
Assessment of GlcNAcase activity and kinetic studies
Both of the purified GlcNAcases were active against p NP-GlcNAc, but Vh Nag2 was found to be much more active than Vh Nag1. We suspect that the full-length Vh Nag1 is expressed as a pro-enzyme, which requires proteolytic processing to attain its full activity. The hydrolysis of p NP-GlcNAc by Vh Nag1 and Vh Nag2 was determined as a function of time (additional file 2, Fig. S1). Vh Nag2 activity was significantly greater than that of Vh Nag1 over the entire course of reaction. In the reaction progress curves, the activity of both enzymes was constant for up to 15 min, and then began to decrease at longer incubation times. Therefore, the reaction time was set to 10 min to ensure that initial velocities were measured in subsequent kinetic experiments.
Kinetic parameters of chitin oligosaccharide hydrolysis.
kcat (s -1 )
kcat /Km (M -1 s -1 )
p NP-GlcNAc (Vh Nag1)
172 ± 48a
p NP-GlcNAc(Vh Nag2)
77 ± 17
179 ± 52
441 ± 98
329 ± 93
496 ± 78
421 ± 76
Time course of chitin oligosaccharide hydrolysis by TLC and HPLC
Kinetic modeling and subsite mapping
where e and c represent the experimental and calculated values, respectively, n is the number of GlcNAc units in the oligosaccharides, and i the reaction time.
The rate constants and the binding free energy changes estimated from the kinetic modeling calculation.
Rate constant (s -1 )
Binding free energy ( k cal mol-1)
The TLC and kinetic data suggested that the catalytic pocket of Vh Nag2 probably contains a sequence of four favorable GlcNAc binding subsites, designated (-1)(+1)(+2)(+3). Such an implication is certainly supported by the binding free energy changes obtained by the kinetic modeling. As shown in Table 2, a very high positive value +7.0 kcal/mol for subsite (-2) suggests that there is a large steric hindrance interfering with binding at this subsite. A less positive value of +0.9 kcal/mol of subsite (+4) suggests that the sugar residue binding to this subsite is moderately unfavorable, but still possible. Thus, GlcNAc4 binds to (-1) (+1), (+2), and (+3) more strongly than GlcNAc5 to (-1) (+1), (+2), (+3) and (+4). A low negative free energy change, -0.1 kcal/mol, was estimated for binding at subsite (+3). This is consistent with the fact that the GlcNAc3 binding affinity to the subsites (-1) (+1), and (+2) (Km = 441 μM) is somewhat weaker than that of the GlcNAc4 binding to (-1) (+1), (+2), and (+3) (Km = 329 μM). All of these results clearly indicate that an array of four GlcNAc binding subsites (-1)(+1)(+2)(+3) define the substrate affinity of Vh Nag2. It appears that the GlcNAcases reported to date possess two major types of substrate specificity. The first type has marked preference towards chitobiose, while the other type favours chitooligomers (GlcNAc3-6) over chitodimer. Examples of the former type are hyperthermophilic kodakaraensis KOD1 GlmATK , Sm Chb , and S. thermoviolacacus NagC . On the other hand, V. furnissii exoI , human di-N-acetylchitobiase , and Vh Nag2 (in this report) are among the other type. The active site of the second type of GlcNAcases has been demonstrated to contain three to five GlcNAc binding subsites, depending on the substrate specificity of individual enzymes. Multiple sugar-binding-site architecture is not uncommon, as has been demonstrated for other exo-glycosidases such as Aspergillus niger cellobiase , a GH-3 enzyme that degrades cellobioase and cello-oligosaccharides into glucose units. The catalytic center of this enzyme has been reported to contain up to five binding subsites. Also, structural studies of the active site of Bacillus halodurans C-125 REX , a GH-8 exo-oligoxylanase that hydrolyzes xylooligosaccharides to xylose from the reducing end, revealed three substrate binding subsites.
This study reports the isolation, cloning and recombinant expression of the genes encoding two intracellular GH-20 GlcNAcases from a marine bacterium, Vibrio harveyi type strain 650. Data obtained from TLC and quantitative HPLC suggested that the active GlcNAcase homolog (Vh Nag2) was an exolytic enzyme that degraded chitin oliogmers, releasing GlcNAc as the end product. Kinetic modeling suggested that the active site of Vh Nag2 comprises four GlcNAc binding subsites, (-1), (+1), (+2), (+3). Such subsite identification is strongly supported by kinetic data, which showed chitin tetramer as the most effective substrate for this enzyme.
Bacterial strains and vectors
V. harveyi type strain 650 was a marine isolate from Greek sea bass and was a gift from Professor Brian Austin, Heriot-Watt University, Edinburgh, United Kingdom. E. coli strain DH5α was used for routine cloning and plasmid preparations. pGEM®-T easy vector used for subcloning purpose was a product of Promega (Promega Pte Ltd, Singapore Science Park I, Singapore). The pQE 60 expression vector and E. coli type strain M15 (Qiagen, Valencia, CA, USA) were used for cloning and a high-level expression of recombinant GlcNAcases.
Cloning of the DNAs encoding VhNag1 and VhNag2
Three sets of oligonucleotide primers were designed based on the three putative GlcNAce genes, designated VIBHAR_01265, VIBHAR_03430, VIBHAR_06345) from V. harveyi type strain ATCC BAA-1116 in the CAZy database. However, only two PCR products, namely VhNag1 and VhNag2, were successfully amplified from the genomic DNA of V. harveyi type strain 650. The oligonucleotides used for amplification of VhNag1 DNA are 5'-AGGATCC GGGCAGGGTAAAATC-3' for the forward primer and 5'-AGGAGATCT ATCGGTTAAAGTGTGAAG-3' for the reverse primer. For VhNag2 DNA, 5'-AGGGATCC GAATACCGTGTTGATTTA-3' was used as the forward primer and 5'-AATAGATCTC TTCCACGGTTTACGGTA-3' for the reverse primer. The PCR products of expected sizes (2.3 kbp for VhNag1 and 1.9 kbp for VhNag2) were cloned in the pQE60 expression vector using Bam H I and Bgl II cloning sites (sequences underlined) following the protocol supplied by the manufacturer.
Nucleotide, amino acid sequence and phylogenic analyzes
The nucleotide sequences of VhNAg1 and VhNag2 were determined by automated double stranded DNA sequencing (Bio Service Unit, Thailand Science Park, Bangkok, Thailand). Ambiguous nucleotides were re-confirmed twice before submission to the Genbank database. The amino acid sequence alignment was constructed using ''CLUSTALW'' algorithm commercially available in Lasergene v.7 (DNASTAR, Inc., WI, USA) and displayed using the Genedoc program (http://www.psc.edu/biomed/genedoc/). The putative sequences of VhNag1 and VhNag2 were aligned with the previously published V. harveyi chitobiase  together with four bacterial and two human GlcNAcases of known structures.
Protein expression and purification
The full-length Vh Nag1 and VhNag2 DNAs were cloned into pQE60 expression vector and expressed in E. coli M15 host as the C-terminally (His)6-tagged polypeptides. The cells were grown at 37°C in Luria Bertani (LB) medium containing 100 μg/ml ampicillin until the OD600 of the cell culture reached 0.6. Expression was induced by the addition of isopropyl thio-β-D-galactoside (IPTG) to a final concentration of 0.5 mM. After 18 h of induction at 20°C, the cell pellet was collected by centrifugation, re-suspended in lysis buffer (20 mM Tris-HCl buffer, pH 8.0, containing 150 mM NaCl, 1 mM phenylmethylsulphonyl fluoride (PMSF), and 1.0 μg/ml lysozyme), and then lysed on ice using a Sonopuls Ultrasonichomogenizer with a 6-mm-diameter probe (50% duty cycle; amplitude setting, 20%; total time, 30 s, 6-8 times). Unbroken cells were removed by centrifugation at 12,000 × g, 20 min at 4οC. The supernatant was immediately applied to a Ni-NTA agarose affinity column (1 × 10 cm) (QIAGEN GmbH, Hilden, Germany), and the chromatography was carried out under gravity at 4°C. The column was washed thoroughly with 5 mM imidazole, followed by 20 mM imidazole in equilibration buffer (20 mM Tris-HCl buffer, pH 8.0), and then 250 mM imidazole in the same buffer. Three eluted fractions (10 ml each) were collected and analyzed by 12% SDS-PAGE  to confirm purity. GlcNAcase fractions were pooled and then subjected to several rounds of membrane centrifugation using Vivaspin-20 ultrafiltration membrane concentrators (Mr 10,000 cut-off, Vivascience AG, Hannover, Germany) for complete removal of imidazole. The final concentration of the protein was determined by Bradford's method .
Confirmation of recombinant expression by mass spectrometry
The purified Vh Nag1 and VhNag2 (2 μg) were applied in parallel onto a 12% SDS-PAGE gel, and stained with Coomassie blue R-250 after electrophoresis. After destaining, protein bands were subjected to in-gel digestion with trypsin (sequencing grade, Promega) using a standard protocol . The resultant peptides were analyzed by high resolution nanoESI/FTMS by the mass spectrometry facility located at the Max-Planck Institute for Molecular Physiology, Dortmund. Data bank searching was performed with ''Mascot search'' (http://www.matrixscience.com/) for peptide mass fingerprinting.
GlcNAcase activity assays
GlcNAcase activity was determined spectrophotometrically using p NP-GlcNAc (Sigma-Aldrich Pte Ltd., The Capricorn, Singapore Science Park II, Singapore) as substrate or by a reducing sugar assay using GlcNAc2-6 (AMS Biotechnology (Europe) Ltd, Oxfordshire, UK) and colloidal chitin as substrates. For the p NP assay, a 100-μl assay mixture contained the protein sample (50 μg), 125 μM p NP-GlcNAc), and 0.065 M phosphate buffer, pH 7.0 The enzymic reaction was continued for 10 min at 37°C before being terminated by the addition of 100 μl 3 M Na2CO3. The amount of p-nitrophenol (p NP) released was determined spectrophotometrically at 405 nm in a microtiter plate reader (Applied Biosystems, Foster City, CA, USA). Molar concentrations of p NP were calculated from a calibration curve constructed with 0-20 nmol p NP. For the reducing sugar assay, the reaction mixture (100 μl) contained 250 μM GlcNAc2-6 in 0.1 M phosphate buffer, pH 7.0 and 200 μg enzyme or 500 μM p NP-glycoside in 0.1 M phosphate buffer, pH 7.0 and 100 μg enzyme. The reaction mixture was incubated at 37°C for 15 min in a Thermomixer comfort (Eppendorf AG, Hamburg, Germany), then heated at 100°C for 10 min. The entire reaction mixture was subjected to 3,5-dinitrosalicylic acid (DNS) assay following the protocol described by Miller . Release of the reducing sugars was detected spectrophotometrically at 540 nm and molar concentrations of the released sugars were estimated using a standard calibration curve of GlcNAc (0-500 nmol). For colloidal chitin, the reaction mixture (200 μl), containing 5% (w/v) colloidal chitin (prepared according to Hsu and Lockwood, 1975 ), 0.1 M phosphate buffer, pH 7.0, and 200 μg enzyme, was incubated at 37°C for 15 min. After centrifugation at 12,000 × g for 1 min to precipitate the remaining chitin, the product concentration in the supernatant (100 μl) was determined by DNS method as described for GlcNAc2-6.
Effects of pH on the enzymatic activity
A discontinuous assay was used to determine the pH profiles of Vh Nag1 and Vh Nag2. The reaction mixtures containing 500 μM p NP-GlcNAc were pre-incubated at 37°C for 5 min at different pH values ranging from 2.5 to 9.0 using the McIlvaine's sodium phosphate-citric acid - KCl buffer system , followed by addition of 1 μg Vh Nag1 or 0.5 μg Vh Nag2. After 10 min of incubation, the reactions were terminated with 100 μl of 3 M Na2CO3. The amounts of p NP released were estimated as described for the p NP assay.
Time course of substrate analysis by thin-layer chromatography
Hydrolysis of chitooligosaccharides (GlcNAc2-6) by Vh Nag2 was carried out in a 20-μl reaction mixture, containing 0.1 M phosphate buffer, pH 7.0, 2.5 mM substrate and 5 μg purified enzyme. The reaction mixture was incubated at 30°C for 1, 5, 10, 15, 30 min, 3 h or 18 h, and the reaction terminated by boiling for 5 min. For product analysis, five 1-μl aliquots of each reaction mixture were applied to a silica TLC plate (7 × 10 cm), and then chromatographed three times (1 h each) in a mobile phase containing n-butanol:methanol:28% ammonia solution:H2O (10:8:4:2) (v/v), followed by spraying with aniline-diphenylamine reagent and baking at 180°C for 3 min. To determine the time course of chitin hydrolysis, the reaction was carried out in a 150-μl reaction mixture, containing 0.1 M phosphate buffer, pH 7.0, 20 mg colloidal chitin, and 50 μg purified enzyme. Subsequent reactions and determination of the reaction products were analyzed by TLC as described for chitooligosaccharide hydrolysis.
Time-course analysis of chitooligosaccharide hydrolysis by HPLC
A reaction mixture (100 μl) containing 1.25 mM chitin oligosaccharide (GlcNAc2-6), 38 μM Vh Nag2 and 0.2 M sodium phosphate buffer, pH 7.0 was incubated at 30°C. An aliquot of 12 μl was transferred to a new microfuge tube containing 12 μl 0.1 M NaOH after 5, 10, 15, 30, 60, 120 and 180 min, and the enzymic reaction was stopped by snap-freezing in liquid N2 and the mixture immediately stored at -20°C. To quantitatively determine the time-course of substrate degradation and product formation, 15-μl of the reaction mixture was applied to a gel-filtration column of TSK-GEL G2000PW (7.5 × 600 mm, Tosoh) connected with a Hitachi L-7000 HPLC system (Hitachi Koki Co., Ltd, Tokyo). Elution was conducted with distilled water at a flow rate of 0.3 ml/min, and the substrate and products were monitored by their absorption at 220 nm. Based on the peak areas obtained from the elution profiles, oligosaccharide concentrations were calculated using a standard curve obtained with authentic saccharide solutions, and then plotted against the reaction time to obtain the reaction time-course.
Kinetic parameters were determined using p NP-GlcNAc and chitooligosaccharides (GlcNAc2-6) by the reducing sugar assay as described above, with 0-500 μM of each substrate in the reaction mixture. The amounts of the reaction products were determined from a standard curve of GlcNAc (0-1.75 μmol). Kinetic parameter values were evaluated from three independent sets of data using the nonlinear regression function obtained from the GraphPad Prism v.5.0 (GraphPad Software Inc., San Diego, CA).
Kinetic modeling of substrate hydrolysis
Kinetic modeling of the reaction time-course obtained by HPLC was carried out using the reaction model reported for the Coccidioides immitis family 18 chitinase . The model scheme is shown in Fig. 7. Considering that the enzyme hydrolyzes the oligosaccharide substrate exolytically from the non-reducing end, Vh Nag2 was assumed to have a (-2)(-1)(+1)(+2)(+3)(+4)-type binding cleft, where subsite (-2) should have an unfavorable (positive) binding free energy change. By assuming rapid binding equilibrium, the concentrations of the ES-complexes formed through the individual binding modes (C n,i , B i,j , and A i ) were calculated from the binding constants, which were obtained from the binding free energy values of individual subsites occupied with the sugar residues assuming additivity. Details of the calculation method were described by Honda and Fukamizo .
- GlcNAcn or NAG:
β 1-4 linked oligomers of N-acetylglucosamine residues where n = 1-6
- p NP-GlcNAc p:
open reading frames
Thin Layer Chromatography.
This work was financially supported by Suranaree University of Technology (Grant no SUT1-102-52-24-08). We are grateful to P. Janning and A. Brockmeyer, Max Planck Institute of Molecular Physiology, Dortmund, for nano-HPLC/ESI-FTMS measurements and associated data interpretation.
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