Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M601783200 on July 24, 2006

J. Biol. Chem., Vol. 281, Issue 41, 30650-30659, October 13, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
281/41/30650    most recent
M601783200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Southall, S. M.
Right arrow Articles by Oubrie, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Southall, S. M.
Right arrow Articles by Oubrie, A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Soluble Aldose Sugar Dehydrogenase from Escherichia coli

A HIGHLY EXPOSED ACTIVE SITE CONFERRING BROAD SUBSTRATE SPECIFICITY*Formula

Stacey M. Southall1, Justin J. Doel, David J. Richardson, and Arthur Oubrie2

From the Centre for Metalloprotein Spectroscopy and Biology, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom

Received for publication, February 24, 2006 , and in revised form, July 24, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
A water-soluble aldose sugar dehydrogenase (Asd) has been purified for the first time from Escherichia coli. The enzyme is able to act upon a broad range of aldose sugars, encompassing hexoses, pentoses, disaccharides, and trisaccharides, and is able to oxidize glucose to gluconolactone with subsequent hydrolysis to gluconic acid. The enzyme shows the ability to bind pyrroloquinoline quinone (PQQ) in the presence of Ca2+ in a manner that is proportional to its catalytic activity. The x-ray structure has been determined in the apo-form and as the PQQ-bound active holoenzyme. The beta-propeller fold of this protein is conserved between E. coli Asd and Acinetobacter calcoaceticus soluble glucose dehydrogenase (sGdh), with major structural differences lying in loop and surface-exposed regions. Many of the residues involved in binding the cofactor are conserved between the two enzymes, but significant differences exist in residues likely to contact substrates. PQQ is bound in a large cleft in the protein surface and is uniquely solvent-accessible compared with other PQQ enzymes. The exposed and charged nature of the active site and the activity profile of this enzyme indicate possible factors that underlie a low affinity for glucose but generic broad substrate specificity for aldose sugars. These structural and catalytic properties of the enzymes have led us to propose that E. coli Asd provides a prototype structure for a new subgroup of PQQ-dependent soluble dehydrogenases that is distinct from the A. calcoaceticus sGdh subgroup.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Proteins that require the prosthetic group pyrroloquinoline quinone (PQQ)3 for catalysis form the largest and best-characterized subclass of quinoproteins, catalyzing the conversion of a wide range of different alcohol- and aldehyde-containing compounds to their corresponding aldehydes/ketones and acids as the first step in the catabolism of these compounds. On the basis of amino acid sequence and three-dimensional structure, these proteins can be divided in two classes. One class, of which methanol dehydrogenase is the classic example, exhibits an eight-bladed beta-propeller structure of ~600 amino acids. The second class includes the structurally defined soluble glucose dehydrogenase (sGdh) from Acinetobacter calcoaceticus, which is a 454-residue protein with a six-bladed beta-propeller domain (1). Putative homologues of sGdh have been identified in phylogenetically diverse prokaryotic genera spanning Bacteria and Archaea (1). A role for PQQ-dependent enzymes in eukaryotes has recently been suggested but is still a matter for debate (2–5).

Until now, the A. calcoaceticus sGdh was the only protein of the second class to have been extensively characterized. This enzyme functions as a dimer of identical 50-kDa subunits (6). Each monomer binds three calcium ions and one PQQ cofactor molecule (7, 8). One of the calcium ions is needed for activation of PQQ, and the others are required for functional dimerization of the protein (8, 9). The protein oxidizes a wide variety of mono- and disaccharides as well as the trisaccharide maltotriose to their corresponding lactones (10, 11). The enzyme has a beta-propeller fold comprising six four-stranded, antiparallel beta-sheets (12). PQQ is bound in a wide solvent-accessible active site in the center of the molecule, near the pseudo-6-fold rotation symmetry axis (8, 13). Structures of complexes with the substrate glucose and competitive inhibitor methylhydrazine have resolved the substrate-binding site and led to elucidation of the catalytic mechanism of the reductive half-cycle (the substrate oxidation process) (8, 13).

The A. calcoaceticus sGdh has been successfully incorporated into successful diagnostic systems to monitor glucose levels in the blood of diabetics (14–16). This system has the advantage of being extremely rapid due to the high catalytic activity of sGdh and of being oxygen-independent. The latter feature sets it apart from systems based on the flavoprotein glucose oxidase, for which the oxidative half-cycle of the protein involves an obligatory reaction with oxygen (17). In many cases the bacterial and archaeal homologues of sGdh identified previously (1) have rather low amino acid sequence identity with the A. calcoaceticus sGdh and thus their specificity and activity toward glucose and maltose cannot be predicted. Determination of the three-dimensional structures of these proteins and comparison with A. calcoaceticus sGdh may allow identification of factors involved in determining the substrate specificity of these proteins and suggest potential protein engineering targets to improve the current diagnostic systems.

Here we report the first expression of the yliI (accession code NP415358) gene of Escherichia coli and purification of its gene product. From bioinformatic analysis, the gene product is predicted to be a PQQ-binding protein that has a low 18% identity with the A. calcoaceticus sGdh. Our biochemical analysis shows this enzyme to have a low affinity for both glucose and maltose but a generic promiscuity toward mono-, di-, and trisaccharide aldose sugars. Structural determination of YliI reveals that the PQQ-binding site lies in a shallow, solvent-exposed cleft on the surface of the protein, providing structural insight into the poor substrate selectivity. We have analyzed the primary sequences of the putative A. calcoaceticus sGdh homologues available in the data base and suggest that functional evolutionary divergence of these proteins has generated distinct subtypes. The significant catalytic and structural differences between the enzymes lead us to propose that the E. coli yliI gene product provides the first structure for a new subtype of soluble PQQ-dependent dehydrogenases, with substrate selectivity and structural features that distinguish them from A. calcoaceticus sGdh enzyme type and which we propose to call soluble aldose sugar dehydrogenase (Asd).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Bacterial Strains, Growth Media, and Recombinant DNA Techniques—E. coli cells were grown at 37 °C in LB liquid cultures or agar plates (18). Kanamycin was used at 25 mg/liter when appropriate. Plasmid DNA was isolated using Qiagen miniprep (Qiagen) kits. Chromosomal DNA was isolated using DNeasy Tissue kits (Qiagen). E. coli cells were transformed using standard methods. PCR was carried out using Pfu (Promega) or Taq (Roche Applied Science) DNA polymerases. Restriction enzymes were purchased from Promega. All constructs obtained were verified by DNA sequence analysis.

Construction of the YliI Expression Plasmid—The yliI expression plasmid pET M-11 yliI was constructed by amplifying a DNA fragment containing the yliI gene (without the first twenty codons encoding the original peptide) from E. coli K12 genomic DNA using PCR with these primers: forward, 5'-CATGCCATGGCTCCTGCAACGGTAAATGTCGAA-3'; reverse, 5'-CGCGGATCCCTAAATGCGTGGGCTAACTTTAAGTAATTC-3'. The product was digested with NcoI and BamHI restriction enzymes and ligated into the pET M-11 vector (a gift from Arie Geerlof and Gunter Stier, European Molecular Biology Laboratory, Heidelberg, Germany) cut with the same enzymes and treated with calf intestinal phosphatase. The plasmid obtained was transformed into competent DH5{alpha} cells and selected on LB plates containing kanamycin. Positive clones were confirmed by DNA sequence analysis.

Expression of E. coli yliI—The pET M-11 yliI construct was transformed into E. coli BL21 (DE3) cells for production of recombinant YliI protein. Single colonies were used to inoculate 5 ml of LB medium containing kanamycin at 37 °C for 16 h. This culture was used to inoculate 500 ml of LB medium containing kanamycin. Cultures were grown until late log phase (A280 nm = 0.8) and then cooled to 25 °C prior to the induction of protein expression with 0.5 mM isopropyl-beta-D-thiogalactopyranoside (IPTG). The cells were maintained at 25 °C for 5 h and then harvested by centrifugation (3000 x g, 20 min, 4 °C).

For production of selenomethionine-containing E. coli YliI, the E. coli selenium auxotroph strain B834 (DE3) was transformed with the plasmid pET M-11 yliI. The transformed B834 (DE3) cells were initially grown at 37 °C on M9 minimal medium, supplemented with 50 µg/ml methionine. When the cells reached an A600 of 1.0, the culture was centrifuged at 6000 x g for 10 min at 4 °C. Cells were resuspended in M9 minimal medium without methionine and incubated with shaking at 37 °C. After 6 h, seleno-L-methionine was added to a final concentration of 50 µg/ml, and the culture was incubated with shaking at 37 °C for 30 min. Overexpression of yliI was then induced by cooling the culture to room temperature and adding IPTG to a final concentration of 1 mM. The culture was incubated with shaking at 25 °C for a further 10 h. Cells were harvested by centrifugation at 7000 x g for 15 min at 4 °C. The incorporation of selenomethionine into purified protein was verified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Purification of E. coli YliI—Harvested cells were washed and resuspended in 20 mM Tris-HCl, 100 mM NaCl, 15 mM MgCl2, pH 7.5. Cell suspension was incubated on ice with DNase for 15 min then sonicated while on ice. Cell debris was pelleted by centrifugation (35,000 x g, 30 min, 4 °C). The soluble fraction obtained after centrifugation was applied to, and eluted from, a hand-poured Ni2+-nitrilotriacetic acid metal chelation affinity column (Amersham Biosciences) according to the manufacturer's instructions. Fractions containing His-tagged YliI were pooled and dialyzed into 20 mM Tris-HCl, 100 mM NaCl, 0.5 mM EDTA, 1 mM dithiothreitol, pH 8.0. The hexahistidine tag was removed by cleavage with tobacco etch virus protease. The cleaved protein was concentrated using an ultrafiltration cell (Amicon) and applied to a S200 Sephadex gel filtration column equilibrated with 20 mM HEPES, 100 mM NaCl, pH 7.5. Fractions containing pure YliI were pooled and concentrated using an ultrafiltration cell (Amicon). Protein expression and purification was monitored by SDS-PAGE using 12% acrylamide Tris-glycine gels. After electrophoresis, the gels were stained with Coomassie Blue. Protein concentrations of extracts and purified recombinant YliI were determined according to the method of Bradford using bovine serum albumin as a standard.

Enzyme Reconstitution with PQQ—In order to reconstitute apo-YliI with PQQ, purified protein (5.45 mg/ml) was incubated with a 10-fold molar excess of PQQ in 20 mM HEPES, 100 mM NaCl, 1 mM CaCl2, pH 7.5, at 4 °C for 16 h. Unbound PQQ was removed by passing the mixture over a Sephadex G-25 column (PD-10; Amersham Biosciences). This step was also used to exchange the protein into 20 mM potassium phosphate, pH 7. To determine the effect that PQQ binding had on YliI activity toward glucose, an assay was set up as follows. 8.6 µl of apo-YliI (5.8 mg/ml), 5 µl of 10 mM CaCl2, and 0–25 µl of 50 µM PQQ were prepared to a final volume of 50 µl with 50 mM potassium phosphate buffered to pH 7. Solutions were incubated for 16 h at 4 °C, and 10 µl was used to reduce 2,6-dichloroindophenol (DCIP) in the presence of 100 mM D-glucose according to the standard assay described below.

The Aldose Sugar Dehydrogenase Assay—Enzyme activity was measured by observing the reduction at 25 °C of DCIP at 600 nm in a UV-visible spectrophotometer (U-3310; Hitachi). The reaction volume was 1 ml, typically containing 50 µM DCIP, 100 mM substrate, 50 mM potassium phosphate buffer, pH 7, and 1–8 µg of holo-YliI. Reduction of DCIP was followed over 150 s, initial rates being estimated from a linear reduction in absorbance at 600 nm (E600 (DCIP) = 21 mM–1 cm–1), typically over the first 20 s of observation. Sugar substrates were prepared 24 h in advance to allow equilibration of anomers. Enzyme activities are expressed in units mg–1, where 1 unit of enzyme activity represents the reaction of 1 µmol of sugar substrate/min. Kinetic constants were estimated by a nonlinear least squares method using MicroMath Scientist. The pH optimum of the holo-YliI was determined in buffers of 50 mM potassium phosphate and 50 mM Bis-Tris propane over the pH ranges 6.0–8.0 and 7.0–9.5, respectively.

Analysis of Reaction Products by HPLC—Reaction mixtures totaling 1 ml were prepared containing 790 µl of 1 mM DCIP, 100 µl of 1 M D-glucose, 100 µl of 50 mM potassium phosphate buffer, pH 7, and 10 µl of reconstituted E. coli YliI (3.45 mg/ml). Control reactions without enzyme and without enzyme or glucose but with 79 µl of 10 mM gluconic acid were also prepared. Reaction mixtures were incubated with shaking at 25 °C for 2 h. The reaction was deemed to have reached completion when the mixture became colorless (i.e. all of the DCIP was reduced). DCIP was then removed by four successive extractions with a 1:2:0.001 solution of chloroform/methanol/hydrochloric acid. 10-µl samples were loaded onto an Ion Pac AS4A SC running at 1 ml/min with an isocratic gradient of 1% 150 mM NaOH. Peaks were detected by ED40 conductivity.

Crystallization of YliI—A newly developed seeding protocol to obtain well diffracting crystals of soluble PQQ-dependent enzymes from a number of sources has been established.4 The YliI protein was crystallized at 16 °C using the hanging drop vapor diffusion method. A protein solution of 3 mg/ml was mixed with an equal volume of precipitant solution (17–22% PEG 6000, 1 mM CaCl2, 120 mM NaCl, 100 mM HEPES, pH 7.0, or 17–22% PEG 6000, 1 mM CaCl2, 120 mM NaCl, 100 mM CHES, pH 9.2) to reach a total volume of 2 µl, to which 1 µl of a seed stock solution was added. The drops were equilibrated against a 1-ml reservoir of precipitant solution.

Soaking of YliI Crystals with PQQ—Crystals were transferred to a stabilizing solution comprising 30% (w/v) PEG 6000, 1 mM CaCl2, 120 mM NaCl, 100 mM HEPES, pH 7.0, or 25% PEG 6000, 1 mM CaCl2, 120 mM NaCl, 100 mM CHES, pH 9.2, containing 1 mM PQQ. Crystals were incubated in this solution for up to 12 h and were subsequently flash-frozen using liquid nitrogen in a solution containing 30% (w/v) PEG 6000, 20% (v/v) ethylene glycol, 1 mM CaCl2, 120 mM NaCl, and 100 mM HEPES, pH 7.0, or 30% (w/v) PEG 6000, 20% ethylene glycol, 1 mM CaCl2, 120 mM NaCl, and 100 mM CHES, pH 9.2.

X-ray Diffraction Data Collection and Analysis—X-ray diffraction data sets were collected to a resolution of 1.5 Å at beam line ID29 of the European Synchrotron Radiation Facility (Grenoble, France). Data were processed and reduced using MOSFLM (20) and programs from the CCP4 suite (21). 5% of the data were set aside to follow progress of refinement using the Rfree factor (22). The final round of refinement was carried out using all of the data.


Figure 1
View larger version (68K):
[in this window]
[in a new window]
  		FIGURE 1.
12% SDS-PAGE of the expression and purification of recombinant YliI (Asd) produced in E. coli. Lane 1, molecular weight markers; lane 2, crude extract of BL21 (DE3) cells carrying the pET M-11 YliI vector after induction with IPTG; lane 3, crude extract of BL21 (DE3) cells carrying the pET M-11 YliI vector; lane 4, His-tagged YliI eluted from a Ni2+-nitrilotriacetic acid-agarose column; lane 5, YliI after incubation with tobacco etch virus protease; lane 6, elution of YliI from S200 gel filtration column. Protein staining was done with Coomassie Blue.

 
Structure Determination and Refinement—The structure of apo-YliI from E. coli was determined by single wavelength anomalous dispersion using the anomalous signal of six incorporated selenium atoms per YliI molecule. Identification of the selenium atom sites and subsequent phasing was carried out using programs from the CCP4 suite (21). The obtained phases allowed for 91% of all amino acids in the asymmetric unit of the apoenzyme to be built automatically by ARP/wARP (23). The structure of holo-YliI was determined by molecular replacement using Molrep using a model of the apoenzyme taken from an early stage in the refinement of that model. The models were completed using alternating rounds of manual model building using Coot (24) and automated refinement using REFMAC (25) and ARP (26). As refinement of the protein models approached completion, phases were calculated from the model. Water molecules were incorporated using ARP, and, in the latter stages of refinement, the models were further improved by applying individual atomic anisotropic B-factor refinement using REFMAC. Throughout refinement, progress was monitored with the aid of an Rfree value calculated with 5% of the data. Upon completion of the models, a final round of refinement using all of the data were used to give a single crystallographic R factor. The stereochemistry and geometry of the final model were judged with PROCHECK (27).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
Expression, Purification, and Biochemical Characterization of E. coli YliI—The E. coli YliI was successfully cloned and expressed in E. coli BL21 (DE3). SDS-PAGE revealed a strong IPTG-inducible band at around 40,000 Da (Fig. 1). The observed molecular mass is consistent with the predicted recombinant protein mass of ~42,000 Da, which decreased to ~39,000 Da after cleavage of the His tag (Fig. 1). A typical purification resulted in pure recombinant YliI as judged by SDS-PAGE with a yield of up to 30 mg of pure protein/liter of culture (Fig. 1). The identity of the pure protein was confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.


Figure 2
View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 2.
UV-visible absorption spectra of E. coli YliI (Asd). A, absorbance spectra of YliI before (i) and after (ii) binding of PQQ. The inset shows reconstitution of YliI with PQQ. Enzyme/cofactor mixes were incubated for 16 h at 4 °C prior to assay in the presence of 100 mM D-glucose and 50 µM DCIP in 50 mM potassium phosphate buffered at pH 7. B, absorbance spectra taken during the titration of YliI with D-glucose. To 0.5 mg/ml holoenzyme in 50 mM potassium phosphate, pH 7 (i), was added successive amounts of 1 M glucose until no further change occurred. Line ii is a scan of fully reduced YliI after the addition of 300 mM D-glucose. All scans were corrected for volume changes.

 
The UV-visible spectrum of apo-YliI is typical of that of a cofactorless enzyme and has a maximum at ~280 nm (Fig. 2). Upon reconstitution with PQQ, a second broader peak corresponding to bound PQQ can be observed, with a maximum at 353 nm (Fig. 2). D-Glucose was selected as a substrate with which to characterize the dependence of activity on PQQ and pH. In the presence of increasing amounts of glucose, the UV-visible spectrum of YliI showed an increase in absorbance corresponding to reduction of PQQ (Fig. 2). The dominant peak at 353 nm increased in size with a new maximum at 338 nm. The activity of apo-YliI toward glucose increased in a linear manner with respect to added PQQ, reaching a plateau at a value of 1 (Fig. 2, inset). From this we deduce that one molecule of PQQ binds per molecule of YliI, similar to that shown with sGdh from A. calcoaceticus (28). CaCl2 was present in these reconstitution assays, and inclusion of the calcium chelator EDTA (1 mM) during reconstitution with PQQ resulted in no activity toward glucose, suggesting that YliI is also a Ca2+-binding protein and that the cation is important for PQQ binding. Once PQQ is bound, the holoenzyme is extremely stable, activity being essentially unchanged after 3 months of storage at 4 °C. The observed oxidation of DCIP to DCIPH2 at 600 nm was shown to be linear with respect to concentration of YliI (data not shown). The products of the D-glucose-dependent enzyme assay were assessed by HPLC. The chromatogram profile was similar to that seen when gluconic acid was applied to the HPLC column (data not shown) and showed no overlap with the chromatograms produced by any of the reaction constituents. This substantiates the theory that E. coli YliI is capable of acting as a D-glucose dehydrogenase, with the reaction product being gluconolactone that is rapidly hydrolyzed in solution to yield gluconic acid.


Figure 3
View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 3.
Comparison of pH dependence upon activity for YliI (Asd) reaction with D-glucose and maltose. A standard assay containing 5 µg of holoenzyme, 50 µM DCIP, and either 100 mM glucose or 100 mM maltose, buffered with 50 mM Bis-Tris propane, was used as described under "Materials and Methods." White squares represent rates observed with maltose (right axis), and black diamonds represent rates observed with glucose (left axis). Points represent the closest value to the mean from three repetitions from which the deviation was less than 5%.

 
The activity of YliI toward glucose was measured over a range of pH using two different buffers, 50 mM potassium phosphate and 50 mM Bis-Tris propane. The activity in the presence of Bis-Tris propane was substantially higher than that observed in potassium phosphate at similar pH. Activity assays were therefore performed in Bis-Tris propane buffer. The activity of YliI toward glucose showed an optimum at pH 8.75 (Fig. 3). This observation is in contrast to that observed with the sGdh from A. calcoaceticus, which has a pH optimum of 7 in potassium phosphate buffer (10). Comparison of the pH profile of YliI for D-glucose with that of maltose reveals substantial differences. In the presence of maltose, the pH activity profile has a maximum of pH 8.0 (Fig. 3). Varying the concentration of D-glucose, maltose, or maltotriose revealed that activity was related to substrate concentration in a manner that approximated Michaelis-Menten kinetics. At pH 8.75, the apparent kinetic parameters for D-glucose were Kcat = 3360 s–1 and Km = 400 mM (Table 1). This is 1 order of magnitude lower than that for A. calcoaceticus sGdh (Table 1) and leads to E. coli YliI having a much lower catalytic efficiency for D-glucose than A. calcoaceticus sGdh (Table 1). The Km values of E. coli YliI for the disaccharide maltose or trisaccharide maltotriose were lower than for glucose, leading to a higher catalytic efficiency for these substrates compared with D-glucose (Table 1). In the case of maltose, this affinity and catalytic efficiency were still much lower than published for A. calcoaceticus (Table 1).


View this table:
[in this window]
[in a new window]
  		TABLE 1
Apparent kinetic parameters for E. coli YliI (Asd) and A. calcoaceticus sGdh with different substrates

Data were collected by observation of rates in the presence of 50 µM DCIP and 50 mM Bis-Tris propane, pH 8.75, as detailed under "Materials and Methods." Parameters were estimated from fitting to the Michaelis-Menten equation. Data are an average of three repetitions for which the deviation was less than 5%. Data for the A. calcoaceticus sGdh are taken from Ref. 11.

 
Determination of the X-ray Crystal Structure of E. coli YliI—The selenomethionine-labeled apoprotein of E. coli YliI crystallized in two different space groups, P21 with two molecules in the asymmetric unit and P212121 with one molecule in the asymmetric unit. Holoenzyme crystals were obtained by soaking crystals of the apoenzyme with a solution containing PQQ. This had no effect on the space group of the crystals (P212121). The model of the apoenzyme was refined at 1.5 Å resolution to a crystallographic R-factor of 14.9% and a free R-factor of 18.3% (Table 2). The model comprises two protein monomers (residues 3–350) and two calcium ions for each molecule in the asymmetric unit, 1085 water molecules, one phosphate molecule, and 25 molecules of ethylene glycol from the cryocoolant. The PQQ-containing model was refined at 1.5 Å to a crystallographic R-factor of 13.2% and a free R-factor of 16.2%. The final model comprised residues 3–350, one molecule of PQQ, 2 calcium ions, 462 water molecules, five sodium ions, one phosphate molecule, one molecule of ethylene glycol, and two molecules of maltose from the cryocoolant bound in the intermolecular spaces.


View this table:
[in this window]
[in a new window]
  		TABLE 2
Data collection and refinement statistics for crystals of E. coli YliI (Asd)

 
Both structures have good overall geometry as judged by PROCHECK, with no residues lying in disallowed regions of the Ramachandran plot (29). 88.7% of residues lie in the most favored regions, with a further 10.9% in additional allowed regions. Two residues consistently lie in the generously allowed region of the Ramachandran plot in both structures: Pro215 and Leu66. Both of these residues are in the cis-peptide conformation. This allows the backbone carbonyls of Gly214 and Pro215 to act as ligands to the active site calcium. The backbone conformation of Leu66 allows it to hydrogen-bond to the OG1 of Thr32 via its carbonyl group, further stabilizing the 2A-2B strand structure. This may be important in maintaining potential hydrogen bonding partners to the substrate, such as Gln62, whose corresponding residue in A. calcoaceticus sGdh, Gln76, is involved in interactions with the glucose O2 hydroxyl.

Overall Structure and PQQ Binding of E. coli YliI—The overall fold of E. coli YliI in both the apoenzyme and the active holoenzyme forms consists of the beta-propeller fold characteristic of this family containing six four-stranded antiparallel beta-sheets (Fig. 4). The PQQ-binding site lies in a shallow, solvent-exposed cleft on the surface of the protein close to the pseudosymmetry axis generated by the six beta-sheets. In contrast to A. calcoaceticus sGdh, the E. coli YliI enzyme crystallizes as a monomer, and this was also confirmed for its native state in solution as determined by gel filtration and dynamic light scattering measurements (data not shown). Each monomer binds two calcium ions, which compares to three in A. calcoaceticus sGdh. In both proteins, one of these calcium ions lies in the PQQ binding pocket and will be discussed later, and another is sandwiched between two of the six strands that make up the propeller fold. It has been shown previously that the propeller fold has a general preference for 7-fold pseudosymmetry (reviewed in Ref. 32). Given that (i) the A. calcoaceticus sGdh and E. coli YliI both contain six blades, (ii) the calcium site is sandwiched between two of the six sheets and thereby part of the structural core, and (iii) four sheets of the A. calcoaceticus sGdh and E. coli YliI structures can be superimposed with low root mean square deviation values on seven-bladed propellers, such as nitrous oxide reductase (30) and quinohemoprotein amine dehydrogenase (31), it seems possible that this conserved calcium site serves to stabilize the propeller structure by allowing six sheets to adopt a stable configuration similar to those adopted by seven-bladed propellers. Moreover, one of the calcium ligands is a conserved glutamate (Glu220) in strand 4C, which may add to the stability of this beta-sheet. The third calcium binding site in A. calcoaceticus also lies in the long loop between strands 4C and 4D, stabilizing the loop in the vicinity of the dimerization interface. This region of the loop is significantly shorter in E. coli YliI and thus does not require the structural stabilization provided by calcium binding (Fig. 4).

The largest, albeit still small, structural change upon binding of PQQ to the enzyme is the rotation of the side chain of His213 to form hydrophobic stacking interactions with PQQ. The corresponding residue in A. calcoaceticus sGdh, Gln246, cannot make such interactions. These hydrophobic contacts might increase the affinity of the enzyme for PQQ and thus increase enzyme stability. There are minor rearrangements of side chains in the active site to facilitate hydrogen bonding interactions between the protein and PQQ (Fig. 5). The side chain of Tyr241 rotates toward PQQ to form hydrogen bonds between the side chain hydroxyl and the O5, O7A, and N6 of PQQ. The side chain of Gln197 is also seen to rotate facilitating additional hydrogen bond formation with PQQ. The structures of the apoand holoenzyme can be overlaid with a root mean square deviation of 0.34 across the main chain atoms.


Figure 4
View larger version (40K):
[in this window]
[in a new window]
  		FIGURE 4.
The overall structure of E. coli YliI (Asd). A, schematic representation of the overall structure of E. coli YliI. "Blades" of the propeller structure are labeled 1–6. B, overlay of the loop region between strands 4C and 4D from E. coli YliI (Asd) (yellow) and A. calcoaceticus sGdh (red), which is involved in dimerization in A. calcoaceticus sGdh. Calcium ions are shown as spheres.

 
The SigmaA-weighted 2Fo – Fc electron density of the PQQ cofactor is well defined, and the quality of the electron density maps allows us to ascertain that the conformation of PQQ is planar (Fig. 5). It was shown that the conformation of oxidized PQQ is not completely planar in the Acinetobacter sGdh (13), whereas the likely reduced state is planar (8). In the E. coli YliI structure, it is clear that PQQ is planar, but it is not possible to be certain of the oxidation state of the enzyme, given that the solvent contains maltose and one turnover thus might have taken place. All protein residues in direct contact with the co-factor have well defined electron density. Comparison of the E. coli YliI structures with those from A. calcoaceticus sGdh (Fig. 6) shows that the geometry of all known participants in the reductive half-reaction (i.e. PQQ, calcium, His127 (His144), and Arg194 (Arg228)) are very similar in the two structures. The charge or hydrogen bonding potential of other key residues, such as Glu146 (Asp163), is maintained. This suggests that the catalytic mechanism for aldose sugar oxidation may be similar.


Figure 5
View larger version (54K):
[in this window]
[in a new window]
  		FIGURE 5.
Stick representation of the PQQ-binding site of E. coli YliI (Asd) in both the apoenzyme and holoenzyme forms. The PQQ cofactor is shown in yellow with surrounding SigmaA-weighted 2Fo – Fc electron density. The hydrogen bonds to the protein from PQQ and the active site calcium ion (red sphere) are shown as dashed red lines. Cyan, apoenzyme; pink, holoenzyme.

 


Figure 6
View larger version (50K):
[in this window]
[in a new window]
  		FIGURE 6.
Stick representation of the active site region of E. coli YliI (Asd) and the glucose-bound form of A. calcoaceticus sGdh. Structures were overlaid using the position of PQQ (orange). Residue labels in parentheses represent the numbering for A. calcoaceticus sGdh. Cyan, YliI (Asd); pink, sGdh.

 
Substrate Specificity of E. coli YliI—The three-dimensional structure of E. coli YliI as described here and the substrate specificity profile of this enzyme have allowed us to understand more about the determinants of substrate specificity in this family of enzymes. Although the key components of the reaction mechanism and a number of other residues with important structural roles in A. calcoaceticus sGdh are conserved in E. coli YliI, not all of the amino acids involved in substrate binding are present, and indeed novel active site features are apparent. The long loop in A. calcoaceticus sGdh between strands 3B and 3C, which contains helix {alpha}2, is significantly shorter in E. coli YliI. This loop contains residues involved in hydrophobic interactions with glucose, such as Leu169, and in hydrogen bonds to glucose, such as Gln168. The hydrophobic residues Tyr343 and Trp346, which are involved in hydrophobic interactions with glucose in A. calcoaceticus sGdh, are also absent in E. coli YliI due to the dramatic difference in structure of the loop between strands 4D and 5A. The significantly shorter loop regions in E. coli YliI generate a very open active site structure with the entire surface of PQQ exposed to solvent (Fig. 7). This imposes little steric hindrance to substrate binding but reduces the interactions that can potentially be made between substrate and enzyme to yield tight binding, which is reflected by the extremely high Km values obtained for glucose and the larger substrates maltose and maltotriose (Table 1). This also prevented us from obtaining structures of E. coli YliI with substrate bound at the active site, despite extensive soaking trials or crystallization in the presence of substrates. Indeed, one substrate, maltose, was used a cryoprotectant, yet maltose molecules did not bind to the active site but rather at other sites on the protein surface.


Figure 7
View larger version (61K):
[in this window]
[in a new window]
  		FIGURE 7.
Representation of the surface charges of YliI (Asd) from E. coli (top) and the D-glucose-bound form of A. calcoaceticus sGdh (bottom). PQQ and D-glucose are shown in yellow. Positively charged regions are colored blue, and negatively charged regions are colored red.

 
The reduced capacity for tight interaction with the glucose and maltose in E. coli YliI as compared with the A. calcoaceticus sGdh (Table 1) led us to explore the relative activity compared with D-glucose of E. coli YliI toward a broader range of aldose sugar substrates (Table 3). Consideration of the chemical nature of the substrates investigated and making the assumption that the substrates bind in a similar orientation to glucose in the A. calcoaceticus sGdh allowed the following conclusions to be made. E. coli YliI, like the A. calcoaceticus sGdh, acts upon the C1-OH of the sugar ring, as shown by the virtual abolition of activity when this group is substituted. This is apparent in the difference in rates between glucose 1-phosphate and glucose 6-phosphate. The YliI enzyme also shows little or no activity toward linear sugars or sugar alcohols as displayed by rates observed with xylitol, inositol, sorbose, and mannitol. The C2-OH functional group is not required for productive binding, as shown by an increase in rate with 2-deoxyglucose and lyxose as substrates. This is not the case for A. calcoaceticus sGdh, which exhibits a ~9-fold decrease in rate for A. calcoaceticus sGdh (11). The amino acids involved in interactions with the C2-OH of glucose in A. calcoaceticus sGdh are completely conserved in E. coli YliI, with the only difference being the distance of Gln62 (Gln76 in A. calcoaceticus) from the putative site of glucose binding. In the superposition, this side chain is positioned at only 1.7 Å from the C2-OH. This implies that it might have to rotate away from the substrate in order to accommodate glucose and most aldose sugar substrates but not 2-deoxyglucose, consistent with the notion that the hydroxyl at the substrate C2 position is not important for binding. In A. calcoaceticus, the inability to form productive hydrogen bonds with C2 of 2-deoxyglucose reduces its relative activity (10). Larger substitutions at the C2 position result in a loss of activity of the E. coli YliI, as indicated by rates observed with glucosamine and N-acetyl glucosamine (Table 3). This illustrates that subtle differences between the protein structures can lead to dramatic changes in reactivity toward certain substrates.


View this table:
[in this window]
[in a new window]
  		TABLE 3
Activity profiles of E. coli YliI (Asd) toward different substrates

The initial substrate concentration was 100 mM in all cases. ND, not detectable. Data are an average of three repetitions for which the deviation was less than 5%.

 
The trend of increasing observed velocity from D-glucose through D-fucose to D-arabinose indicates that the C5 hydroxymethyl extension is not necessary for catalysis, and indeed, its absence enhances activity. According to our super-position of the E. coli YliI and A. calcoaceticus sGdh structures, this extension should not make any productive interactions with the protein or PQQ, and its absence may allow hydrophobic interaction with the E. coli-specific Tyr241. Similar rates observed with D- and L-arabinose indicate no preference for different enantiomers. This is in contrast with sGdh from A. calcoaceticus, which showed an absolute preference for L-arabinose over D-arabinose. This may be due to the loss of hydrogen bonds with the C2-OH and the proximity of Leu169, which is absent in E. coli YliI.

Comparing monosaccharides and disaccharides, maltose (4-O-{alpha}-D-glucopyranosyl-D-glucose), cellobiose (4-O-beta-D-glucopyranosyl-D-glucose), and melibiose (6-O-{alpha}-D-galactopyranosyl-D-glucose) all show rates significantly faster than that with glucose. Maltotriose (O-{alpha}-D-glucopyranosyl-(1–4)-O-{alpha}-D-glucopyranosyl-(1–4)-D-glucose), a trisaccharide, also showed an elevated rate when compared with glucose. This pattern is not observed in A. calcoaceticus sGdh and may reflect the more solvent-exposed active site that provides less steric hindrance to these more bulky sugars.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
In this study, we have characterized a novel PQQ-dependent enzyme from E. coli in terms of its three-dimensional structure, cofactor binding features, and substrate specificity. A large number of other homologues of this protein exist in a phylogenetically wide range of prokaryotes and remain as yet unstudied (1). Phylogenetic analysis of the primary structure of putative homologues available in the data base using BLAST (32), ClustalW (33) and TreeView (19) suggests distinct evolutionary subclasses that cannot be correlated with 16 S ribosomal evolutionary relationships (supplemental Fig. 1). In terms of their primary structure, E. coli YliI and A. calcoaceticus sGdh are only distantly related within this group. The sGdh from A. calcoaceticus shows significantly greater identity (40%) with the 50-kDa PQQ-binding domain of soluble L-sorbosone dehydrogenase from Ketogulonicigenium vulgare (34, 35) than it does with E. coli YliI (18%). The elongated loop regions found in A. calcoaceticus sGdh are also present in K. vulgare L-sorbosone dehydrogenase but are not found in any other homologues identified so far. This underlies a difference size between the sGdh/L-sorbosone dehydrogenase subgroup (~50 kDa) compared with the E. coli YliI subgroup (~40 kDa). The key residues believed to be involved in the reaction mechanism, however, remain conserved across all of the homologues. Significant differences remain between these other homologues, not least in the residues potentially binding to substrate molecules in the active site. Given that the physiological role of all of these proteins is unknown, it is conceivable that they have different functions in these very different organisms. The structure and enzymatic properties of the E. coli PQQ-dependent enzyme have provided insight into what appears to be the common overall structure of this family of soluble quinoproteins but have identified a new subtype with broad substrate specificity toward aldose sugars and unique structural features. Since it has a low affinity for glucose and it is not at all clear that this is the enzyme's physiological substrate, we feel it would be mis-leading to refer to this enzyme as a soluble glucose dehydrogenase and propose that it is henceforth given the more generic name aldose sugar dehydrogenase (Asd).

The E. coli Asd (YliI) is predicted to be part of a periplasmic electron transfer system that can serve to input electrons into the respiratory network. It represents a second example of a PQQ-dependent dehydrogenase that can input sugar-derived electrons into the E. coli respiratory network. The existence of the first system, an integral membrane glucose-ubiquinone oxidoreductase, has been recognized for some time. This enzyme has a Km for D-glucose of about 4 mM (36), 2 orders of magnitude lower than that reported here for the soluble Asd. The paradox of expressing genes encoding two different PQQ enzymes is that E. coli lacks the genetic information required to synthesize the PQQ cofactor. It is, however, chemotactic toward PQQ (41) and thus could use environmental PQQ released by other organisms to activate periplasmic (Asd) or periplasmic facing (membrane glucose-ubiquinone oxidoreductase) apoenzymes. Many of the environmental niches that E. coli and other enteric bacteria with Asd can occupy will have aldose sugars present, and thus this sGdh could provide for bioenergetic options in such environments that further highlight the flexibility of its respiratory network. There has been significant progress in structurally defining this respiratory network in recent years with structures of nitrite reductase, membrane-bound nitrate reductase, formate dehydrogenase, fumarate reductase, succinate dehydrogenase, and cytochrome bo oxidase all emerging (37–43). The present study expands this molecular definition of the E. coli respirome through the determination of the structure of a hitherto unstudied respiratory enzyme.


   FOOTNOTES
 
* This work was supported by Diabetes UK Grant BDA:RD04/0002927. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The atomic coordinates and structure factors (code 2G8S) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1. Back

2 Present address: N.V. Organon, Molecular Design and Informatics, P.O. Box 20, 5340 BH Oss, The Netherlands. Back

1 To whom correspondence should be addressed. E-mail: Stacey.Southall{at}icr.ac.uk.

3 The abbreviations used are: PQQ, pyrroloquinoline quinone; sGdh, soluble glucose dehydrogenase; Asd, aldose sugar dehydrogenase; IPTG, isopropyl-beta-D-thiogalactopyranoside; DCIP, 2,6-dichloroindophenol; CHES, 2-(cyclohexylamino)ethanesulfonic acid; PEG, polyethylene glycol; HPLC, high pressure liquid chromatography; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. Back

4 Sanchez-Weatherby, J., Southall, S. M., and Oubrie, A. (2006) Acta Crystallogr. Sect. F Struct. Biol. Crystalliz. Comm. 62, 518–521. Back


   ACKNOWLEDGMENTS
 
We thank Arie Geerlof and Gunter Stier (EMBL, Heidelberg) for the gift of the pET M-11 vector, Dr. Charles Brearley for help with HPLC analysis, Ann Reilly for production of samples of E. coli Asd (YliI), Dr. Frank Sargent for helpful comments on the manuscript, and Professor Robert Eisenthal (University of Bath) for helpful discussions.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 

  1. Oubrie, A. (2003) Biochim. Biophys. Acta 1647, 143–151[Medline] [Order article via Infotrieve]
  2. Kasahara, T., and Kato, T. (2005) Nature 433, E11–E12
  3. Kasahara, T., and Kato, T. (2003) Nature 422, 832[CrossRef][Medline] [Order article via Infotrieve]
  4. Felton, L. M., and Anthony, C. (2005) Nature 433, E10–E12[Medline] [Order article via Infotrieve]
  5. Rucker, R., Storms, D., Sheets, A., Tchaparian, E., and Fascetti, A. (2005) Nature 433, E10–E12[Medline] [Order article via Infotrieve]
  6. Olsthoorn, A. J. J., and Duine, J. A. (1996) Arch. Biochem. Biophys. 336, 42–48[CrossRef][Medline] [Order article via Infotrieve]
  7. Olsthoorn, A. J. J., Otsuki, T., and Duine, J. A. (1997) Eur. J. Biochem. 247, 659–665[Medline] [Order article via Infotrieve]
  8. Oubrie, A., Rozeboom, H. J., Kalk, K. H., Olsthoorn, A. J. J., Duine, J. A., and Dijkstra, B. W. (1999) EMBO J. 18, 5187–5194[CrossRef][Medline] [Order article via Infotrieve]
  9. Olsthoorn, A. J. J., and Duine, J. A. (1998) Eur. J. Biochem. 255, 255–261[Medline] [Order article via Infotrieve]
  10. Olsthoorn, A. J. J., and Duine, J. A. (1998) Biochemistry 37, 13854–13861[CrossRef][Medline] [Order article via Infotrieve]
  11. Igarashi, S., Hirokawa, T., and Sode, K. (2004) Biomol. Eng. 21, 81–89[CrossRef][Medline] [Order article via Infotrieve]
  12. Oubrie, A., Rozeboom, H. J., Kalk, K. H., Duine, J. A., and Dijkstra, B. W. (1999) J. Mol. Biol. 289, 319–333[CrossRef][Medline] [Order article via Infotrieve]
  13. Oubrie, A., Rozeboom, H. J., and Dijkstra, B. W. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 11787–11791[Abstract/Free Full Text]
  14. Hoenes, J., and Unkrig, V. (1996) U. S. Patent 5,484,708
  15. Tang, Z., Louie, R. F., Lee, J. H., Lee, D. M., Miller, E. E., and Kost, G. J. (2001) Crit. Care Med. 29, 1062–1070[CrossRef][Medline] [Order article via Infotrieve]
  16. Solnica, B., Naskalski, J. W., and Sieradzki, J. (2003) Clin. Chim. Acta 331, 29–35[CrossRef][Medline] [Order article via Infotrieve]
  17. Pazur, J. H., and Kleppe, K. (1964) Biochemistry 35, 578–583
  18. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY
  19. Page, R. D. (1996) Comput. Appl. Biosci. 12, 357–358[Free Full Text]
  20. Leslie, A. G. W. (1992) Joint CCP4 + ESF-EAMCB Newsletter on Protein Crystallography 26
  21. Collaborative Computational Project 4 (1994) Acta Crystallogr. Sect. D Biol. Crystallogr. 50, 760–763[CrossRef][Medline] [Order article via Infotrieve]
  22. Brunger, A. T. (1997) Macromol. Crystallogr. 277, 366–396[CrossRef]
  23. Perrakis, A., Harkiolaki, M., Wilson, K. S., and Lamzin, V. S. (2001) Acta Crystallogr. Sect. D Biol. Crystallogr. 57, 1445–1450[CrossRef][Medline] [Order article via Infotrieve]
  24. Emsley, P., and Cowtan, K. (2004) Acta Crystallogr. Sect. D Biol. Crystallogr. 60, 2126–2132[CrossRef][Medline] [Order article via Infotrieve]
  25. Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Acta Crystallogr. D Biol. Crystallogr. 53, 240–255[CrossRef][Medline] [Order article via Infotrieve]
  26. Lamzin, V. S., and Wilson, K. S. (1997) Methods Enzymol. 277, 269–305[Medline] [Order article via Infotrieve]
  27. Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) J. Appl. Crystallogr. 26, 283–291[CrossRef]
  28. Dokter, P., Frank Jzn, J., and Duine, J. A. (1986) Biochem. J. 239, 163–167[Medline] [Order article via Infotrieve]
  29. Ramachandran, G. N., and Sasisekharan, V. (1968) Adv. Protein Chem. 23, 283–438[Medline] [Order article via Infotrieve]
  30. Haltia, T., Brown, K., Tegoni, M., Cambillau, C., Saraste, M., Mattila, K., and Djinovic-Carugo, K. (2003) Biochem. J. 369, 77–88[CrossRef][Medline] [Order article via Infotrieve]
  31. Satoh, A., Kim, J. K., Miyahara, I., Devreese, B., Vandenberghe, I., Hacisalihoglu, A., Okajima, T., Kuroda, S., Adachi, O., Duine, J. A., Van Beeumen, J., Tanizawa, K., and Hirotsu, K. (2002) J. Biol. Chem. 277, 2830–2834[Abstract/Free Full Text]
  32. Altschul, S. F., Madden, T. L., Schiffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J. (1997) Nucleic Acids Res. 25, 3389–3402[Abstract/Free Full Text]
  33. Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T. J., Higgins, D. G., and Thompson, J. D. (2003) Nucleic Acids Res. 31, 3497–3500[Abstract/Free Full Text]
  34. Miyazaki, T., Sugisawa, T., and Hoshino, T. (2006) Appl. Environ. Microbiol. 72, 1487–1495[Abstract/Free Full Text]
  35. Sugisawa, T., Miyazaki, T., and Hoshino, T. (2005) Biosci. Biotechnol. Biochem. 69, 659–662[CrossRef][Medline] [Order article via Infotrieve]
  36. Dewanti, A. R., and Duine, J. A. (1998) Biochemistry 37, 6810–6818[CrossRef][Medline] [Order article via Infotrieve]
  37. Jormakka, M., Richardson, D., Byrne, B., and Iwata, S. (2004) Structure 12, 95–104[Medline] [Order article via Infotrieve]
  38. Bertero, M. G., Rothery, R. A., Palak, M., Hou, C., Lim, D., Blasco, F., Weiner, J. H., and Strynadka, N. C. (2003) Nat. Struct. Biol. 10, 681–687[CrossRef][Medline] [Order article via Infotrieve]
  39. Bamford, V. A., Angove, H. C., Seward, H. E., Thomson, A. J., Cole, J. A., Butt, J. N., Hemmings, A. M., and Richardson, D. J. (2002) Biochemistry 41, 2921–2931[CrossRef][Medline] [Order article via Infotrieve]
  40. Jormakka, M., Tornroth, S., Byrne, B., and Iwata, S. (2002) Science 295, 1863–1868[Abstract/Free Full Text]
  41. Iverson, T. M., Luna-Chavez, C., Cecchini, G., and Rees, D. C. (1999) Science 284, 1961–1966[Abstract/Free Full Text]
  42. Yankovskaya, V., Horsefield, R., Tornroth, S., Luna-Chavez, C., Miyoshi, H., Leger, C., Byrne, B., Cecchini, G., and Iwata, S. (2003) Science 299, 700–704[Abstract/Free Full Text]
  43. Abramson, J., Riistama, S., Larsson, G., Jasaitis, A., Svensson-Ek, M., Laakkonen, L., Puustinen, A., Iwata, S., and Wikstrom, M. (2000) Nat. Struct. Biol. 7, 910–917[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J DAIRY SCIHome page
T. Kiryu, T. Kiso, H. Nakano, K. Ooe, T. Kimura, and H. Murakami
Involvement of Acetobacter orientalis in the production of lactobionic acid in Caucasian yogurt ("Caspian Sea yogurt") in Japan
J Dairy Sci, January 1, 2009; 92(1): 25 - 34.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
281/41/30650    most recent
M601783200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Southall, S. M.
Right arrow Articles by Oubrie, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Southall, S. M.
Right arrow Articles by Oubrie, A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement