Clonis1 1 Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, Greece; 2 Embrapa Recursos Gene´ticos e Biotecnologia, Brası´lia,
Trang 1Galactosyl-mimodye ligands for Pseudomonas fluorescens
b-galactose dehydrogenase
Design, synthesis and evaluation
C F Mazitsos1, D J Rigden2, P G Tsoungas3and Y D Clonis1
1 Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, Greece;
2
Embrapa Recursos Gene´ticos e Biotecnologia, Brası´lia, Brazil;3Department of Pharmaceutical and Biological Chemistry,
School of Pharmacy, University of London, UK
Protein molecular modelling and ligand docking were
employed for the design of anthraquinone
galactosyl-bio-mimetic dye ligands (galactosyl-mimodyes) for the target
enzyme galactose dehydrogenase (GaDH) Using
appro-priate modelling methodology, a GaDH model was build
based on a glucose-fructose oxidoreductase (GFO) protein
template Subsequent computational analysis predicted
chimaeric mimodye-ligands comprising a
NAD-pseudomi-metic moiety (anthraquinone diaminobenzosulfonic acid)
and a galactosyl-mimetic moiety (2-amino-2-deoxygalactose
or shikimic acid) bearing an aliphatic linker molecule In
addition, the designed mimodye ligands had an appropriate
in length and chemical nature spacer molecule via which
they can be attached onto a chromatographic support
without steric clashes upon interaction with GaDH
Fol-lowing their synthesis, purification and analysis, the ligands
were immobilized to agarose The respective affinity adsor-bents, compared to other conventional adsoradsor-bents, were shown to be superior affinity chromatography materials for the target enzyme, Pseudomonas fluorescens b-galactose dehydrogenase In addition, these mimodye affinity adsor-bents displayed good selectivity, binding low amounts of enzymes other than GaDH Further immobilized dye-lig-ands, comprising different linker and/or spacer molecules, or not having a biomimetic moiety, had inferior chromato-graphic behavior Therefore, these new mimodyes suggested
by computational analysis, are candidates for application in affinity labeling and structural studies as well as for purifi-cation of galactose dehydrogenase
Keywords: affinity chromatography; biomimetic ligands; galactose dehydrogenase; molecular modelling; triazine dyes
Galactose dehydrogenase (GaDH; D-galactose: NAD+
1-oxidoreductase; EC 1.1.1.48) catalyses the
dehydrogena-tion of b-D-galactopyranose in the presence of NAD+to
D-galacto-1,5-lactone and NADH, acting on the C1
posi-tion of the sugar substrate The enzyme generally shows no
absolute specificity either for NAD+, as NAD P+is also
used, albeit to a lesser degree Nor is the enzyme specific for
D-galactose, asD-fucose is a better substrate, although other
sugars (e.g L-arabinose, 2-deoxy-D-galactose) are less
reactive The kinetic mechanism is ordered Bi-Bi, with the
NAD+ binding first to the enzyme [1] GaDH from
Pseudomonas fluorescensis the best studied example, as it
has been cloned and expressed in Escherichia coli [2] and its full nucleotide sequence determined [3] The active macro-molecule possesses two binding sites [4] and consists of two identical subunits each of 33 kDa (304 amino-acid residues) [3] GaDH from Pseudomonas saccharophila has been studied to a lesser extent [5], whereas the enzyme has been identified in plants (e.g green peas, oranges and Arabidopsis thaliana), algae (e.g Iridophycus flaccidum) and several mammals including humans No information is available regarding the catalytic mechanism of GaDH, and its structure has not been determined experimentally or modelled
GaDH is an important analytical tool as at alkaline pH the product galactonolactone is hydrolysed, so that the reaction becomes irreversible The enzyme is therefore useful for the determination of b-D-galactose and a-D-galactose, after the latter is converted to the former
by the application of exogenous mutarotase GaDH is also exploited for the determination of lactose; the milk sugar is hydrolysed by lactase, coupled to GaDH which acts on the resulting b-D-galactose Despite the utility of GaDH, a simple and rapid purification method is not available
The ability to combine knowledge of X-ray crystallo-graphic studies, NMR and homology structures with defined or combinatorial chemical synthesis and advanced computational tools has made rational design of affinity ligands more feasible, powerful, logical and faster [6] In the present work, rigorous protein molecular modelling was
Correspondence to Y D Clonis, Laboratory of Enzyme Technology,
Department of Agricultural Biotechnology, Agricultural
University of Athens, 75 Iera Odos Street, GR-11855 Athens,
Greece Fax: + 30 210 5294307, Tel.: + 30 210 5294311,
E-mail: clonis@aua.gr
Abbreviations: ADH, alcohol dehydrogenase; BM, biomimetic ligand
or mimodye ligand; CB3GA, Cibacron blue 3GA; GaDH, galactose
dehydrogenase; GaO, galactose oxidase; GFO, glucose-fructose
oxidoreductase; GlDH, glucose dehydrogenase; GlO, glucose oxidase;
VBAR, Vilmafix Blue A-R; CDI, 1,1¢-carbonyldiimidazole.
Enzymes: galactose dehydrogenase (GaDH; D -galactose: NAD+
1-oxidoreductase; EC 1.1.1.48).
(Received 31 May 2002, revised 16 August 2002,
accepted 28 August 2002)
Trang 2used to create an objectively sound model of GaDH using as
the best available template glucose-fructose oxidoreductase
(GFO) This model was then exploited in the design of novel
galactosyl-biomimetic chlorotriazine dye-ligands (mimodye
ligands) with bifunctional or chimaeric characteristics In
particular, these galactosyl-mimodye ligands are designed to
bear a structural portion that interacts with the NAD+
-binding site and a biomimetic moiety that interacts with the
sugar-binding site of GaDH The effectiveness of the
bifunctional (chimaeric) ligand concept has been previously
demonstrated with ketocarboxyl- [7,9] and
glutathionyl-biomimetic [10] ligands but never with sugar ones These
mimodye ligands are expected to become useful tools for the
identification of amino-acid residues of the binding sites of
GaDH after affinity labelling For this purpose, the
galactosyl-mimodyes were designed to bear a reactive
chloro-triazine structural scaffold, present in all reactive
triazinyl-dye ligands including the archetypal CB3GA and
VBAR Other mimodyes and certain conventional triazine
dyes are known to act as affinity labels due to their
chlorine(s) atom(s) which react with appropriate residues
of the targeted enzyme active site [11–13] Furthermore,
when the chlorine was substituted with a carefully chosen
spacer molecule, a nonreactive biomimetic ligand was
obtained which could be immobilized on a chromatography
support We envisage that these immobilized ligands will be
of great use in the purification of GaDH from different
sources
E X P E R I M E N T A L P R O C E D U R E S
Materials
b-Galactose dehydrogenase (EC 1.1.1.48, P fluorescens
gene expressed in E coli), galactose oxidase crude
lyophi-lized powder (EC 1.1.3.9, from Dactylium dendroides),
glucose oxidase crude lyophilized powder (EC 1.1.3.4,
from Aspergillus niger, crude), D(+)-galactosamine
(2-amino-2-deoxy-D-galactopyranose; chondrosamine),
D(+)-galactose (minimum 99%),D(+)-glucose,
1,3-diamino-2-hydroxypropane, bromoacetic acid
N-hydroxysuc-cinimide ester, e-amino-n-caproic acid, ethylene-diamine,
1,5-diaminopentane, 1,6-hexane-diamine,
1,12-diaminodo-decane, 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide
(EDAC), 1,1¢-carbonyldiimidazole, o-tolidine,
o-dianisi-dine, lipophilic Sephadex LH-20, CM–Sepharose CL-6B
and DEAE–Sepharose CL-6B were obtained from Sigma
(St Louis, MO, USA) All other diaminoalkanes were
obtained from Aldrich (USA), whereas, shikimic acid was
obtained from Fluka (USA) Peroxidase (from horseradish,
grade I), NAD+ (crystallized lithium salt c 100%) and
crystalline bovine serum albumin (fraction V) were obtained
from Boehringer Mannheim (Germany) Hexylamine and
nutrient broth (for microbiology) were obtained from
Merck (Germany) The agarose chromatography gel
Sepharose CL-6B was obtained from Pharmacia F324
P fluorescens biovar V1 was kindly donated by G J
Nychas (Laboratory of Microbiology and Biotechnology
of Foods, Agricultural University of Athens) Baker’s
yeast, green peas and rabbit liver were purchased at the
local market Glucose dehydrogenase was extracted from
P fluorescens and baker’s yeast, while alcohol
dehydro-genase was extracted from baker’s yeast and green peas
Protein modelling Fold recognition methods [14–17] were employed to deter-mine the best template to use for construction of a model of GaDH Given the low sequence identity between GaDH and the GFO template used (17%) a rigorous modelling strategy was used, as previously (e.g [18,19]) In this way the challenge of modelling based on low sequence identity was met with a strategy designed to maximize model accuracy Although errors will undoubtedly remain, the probability of producing a useful model is thereby enhanced The essential elements of this strategy are the construction and analysis of multiple models (20 in this case), derived from limited randomization of initial coordinates and made with the programMODELLER [20], followed by analysis of packing and solvent exposure characteristics withPROSAII [21] The resulting profiles showed regions of unusual protein struc-ture characteristics as peaks attaining positive values These regions may result from locally inaccurate target-template alignment so that variant alignments, altered in these doubtful regions, were tested through further cycles of model construction and analysis When betterPROSAresults were obtained for the variant alignment it was assumed to
be more correct than the original Stereochemical analysis usingPROCHECKwas also employed, particularly when the optimal target-template alignment had been reached Pro-tein models were visualized using O [22] Structurally similar proteins to the template were sought in the FSSP database (http://www.ebi.ac.uk/dali/fssp) [23].STRIDE [24] was used for the definition of secondary structure
Ligand design and docking The ideal biomimetic would combine moieties that bind both to the cofactor NADand the substrate binding sites The initially considered building elements were two com-mercially available compounds: (a) anthrquinone-diamino-benzosulfonyl-dichlorotriazine (Vilmafix blue A-R or VBAR) containing three of the four ring systems of the well known dye Cibacron Blue 3GA (CB3GA), both known binding mimics of NAD(P) [8,9,25] and (b) 2-amino-2-deoxygalactose, a substrate of GaDH [1] Both these molecules have readily modifiable chemical groups to which could be attached an appropriate linker molecule in order
to effect their fusion 1-Amino-1-deoxygalactose, although commercially available, was not considered as GaDH attacks at the C1 position of the substrate, so that this position was thought better preserved in the ligand However, in place of galactose shikimic acid was considered which, although only moderately structurally similar to GaDH substrates, has a clear advantage over them in terms
of chemical stability Finally, a spacer molecule of appro-priate length and chemical nature was designed to chemi-cally attach the complete ligand, via its triazine group (ring 3), to the chromatographic matrix
The HIC-UP database of heterocompounds [26] was used as a source of the Cibacron Blue-derived, b-D-galactose and shikimic acid components These were rotated and translated with respect to the protein model using O[22] until optimal steric and chemical complementarity was reached The tendency of Cibacron Blue-like ring systems
to bind in NAD(P) binding sites with anthraquinone mimicking adenine, along with biochemical data regarding
Trang 3sugar binding to related enzymes provided useful
informa-tion to guide the docking, as described later Side chain
reorientations to rotameric conformations were allowed
where they significantly enhanced interactions with ligands
The mimodye ligands (e.g BM1 and BM2) were
mod-elled through the fusion of their respective enzyme-bound
components and the resulting complexes refined usingCNS
[27] Topology and parameter files for energy minimization
of the ligand were generated usingXPLO2D[28] and
hand-edited to reflect ideal stereochemical values
Synthesis and purification of the dye-ligands
Amino-alkyl-VBAR dyes (Table 1, structures
aVBAR-fVBAR) Solid commercial VBAR (50 mg, 0.045 mmol
dichloroform, purity 61.3%, w/w) was added to cold water
(2 mL) and the solution was slowly introduced under
stirring to a solution (3 mL) of the alkyl-diamines
(0.73 mmol) The pH was adjusted to 8.9–9.0 and kept at
this value with NaOH (0.1M) until the end of the reaction
(2.5–3 h, 25C) The progress of each reaction was
monitored by TLC
(1-butanol-2-propanol-ethylacetate-wate, 2 : 4 : 1 : 3 v/v/v/v) upon completion of the reaction,
solid NaCl was added (final content 3%, w/v) and the
mixture was left at 4C The pH of the mixture was
adjusted with HCl (1M) to 1.0 and the precipitate was
filtered (Whatman paper filter 50, hardened), washed with
5 mL each of HCl (1M) and cold acetone, then with 7 mL
of diethyl ether and dried under reduced pressure The solid dye (approximately 30 mg) was dissolved in 50 : 50 water/ methanol (50%) and dimethylsulfoxide (50%) mixture, and purified on a lipophilic Sephadex LH-20 column (30· 2.5 cm) [29] The purified product was stored in a desicator at 4C
Hydrophilic spacer-VBAR dye (Table 1, structure gVBAR; Fig 1) Stage 1: solid commercial VBAR (20 mg, 0.018 mmol dichloroform, purity 61.3%, w/w) was added to cold water (1 mL) and the solution was slowly introduced under stirring to 1,3-diamino-2-hydroxypropane (3 mL, 0.29 mmol) The pH was adjusted to 8.9–9.0 and kept at this value with NaOH (0.1M) until the end of the reaction (2.5–3 h, 25C) The progress of the reaction was monitored by TLC (1-butanol-2-propanol-ethylacetate-water, 2 : 4 : 1 : 3 v/v/v/v) Upon completion of the reaction, the dye was purified according to the method already described (see above) Stage 2: the purified product, 1,3-diamino-2-hydroxypropano-VBAR, was dissolved in dimethylsulfoxide/water (3 mL, 50 : 50, v/v) and the pH was adjusted to 7.5 with NaOH (0.1M) 0.2 mmol of bromoacetic acid N-hydroxysuccinimide ester [30,31] were dissolved in dioxane (1 mL) and this solution was
Table 1 The structures of amino-alkyl-VBAR dyes (a-fVBAR),hydrophilic spacer-VBAR dye (gVBAR),galactosamine-VBAR dye and archetypal VBAR dye.
a
Following ligand immobilization, the -NH 2 group has replaced the -Cl atom.bThe galactosamine-VBAR dye was synthesized employing the procedure for amino-alkyl-VBAR dyes but using the amino-sugar instead the diamino-alkane.
Trang 4introduced to the dye solution The pH was maintained to
7.5 until the end of the reaction (1.5 h, 4C, as judged by
TLC) The progress of the reaction was monitored by TLC
(1-butanol-2-propanol-ethylacetate-water, 2 : 4 : 1 : 3 v/v/
v/v) Upon completion of the reaction, the mixture was
lyophilized and the dye was purified on the lipophilic
Sephadex LH-20 column [29] Stage 3: the purified product,
bromoacetylated 1,3-diamino-2-hydroxypropano-VBAR,
was dissolved in 0.1M NaHCO3, pH 9.0 (2 mL) and the
solution was slowly introduced under stirring to a solution
of 0.4M1,3-diamino-2-hydroxypropane in 0.1MNaHCO,
pH 9.0 (2 mL), while maintaining the pH to 9.0 with HCl (1M) The solution was then left under stirring for another 48–72 h (25C), without further adjustment of the pH The progress of the reaction was monitored by TLC (1-butanol-2-propanol-ethylacetate-water, 2 : 4 : 1 : 3 v/v/v/v) Upon completion of the reaction, the dye was purified according
to the method already described (see above)
Biomimetic dye BM1 (Table 2, structure BM1; Fig 1) Stage 1: purified hydrophilic spacer-VBAR, structure g (approx 15 mg, 0.017 mmol) was dissolved in dimethyl-sulfoxide/water (3 mL, 50 : 50, v/v) and the solution was introduced under stirring to e-amino-n-caproic acid (2 mL, 0.17 mmol) The pH was adjusted to 9.0 and the mixture was left shaking at 60C for 3 h The progress of the reaction was monitored by TLC (1-butanol-2-propanol-ethylacetate-water, 2 : 4 : 1 : 3 v/v/v/v) Upon completion of the reaction, the dye was purified according to the method already described (see above) Control dye C6gVBAR (Table 2) was synthesized in the same way Stage 2: the purified product obtained from stage 1, was dissolved in dimethylsulfoxide/water (3 mL, 50 : 50, v/v), introduced to
a solution ofD(+)-galactosamine (3 mL, 0.62 mmol), and the pH was adjusted to 4.6, before freshly prepared solution
of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (0.3 mL,
250 mg) was introduced dropwise under stirring over a period of 5 min, while maintaining the pH at 4.6–5.0 The reaction was stirred for 20 h at 25C without pH adjustment and monitored by TLC (1-butanol-2-propanol-ethylacetate-water, 2 : 4 : 1 : 3 v/v/v/v) A silver nitrate ammonia solution was used as a spray reagent for detecting the galactose-analogue in the newly synthesized dye [32] The product, structure BM1, was precipitated by addition
of solid NaCl (final content 15%, w/v), filtered and washed with 7 mL of NaCl solution (15%, w/v) and 5 mL of cold acetone, and dried under reduced pressure The product was re-suspended in 2 mL of water and precipitated by addition
of solid NaCl (final content 10%, w/v) The precipitate was filtered and washed with 7 mL each of NaCl solution (10%, w/v) and cold acetone, desiccated with 7 mL of diethyl ether and dried under reduced pressure
Biomimetic dye BM2 (Table 2, structure BM2; Fig 2) Stage 1: solid commercial VBAR (20 mg, 0.018 mmol dichloroform, purity 61.3%, w/w) was added to cold water (1 mL) and the solution was slowly introduced under stirring to a solution (3 mL) of 1,3-diaminopropane (0.29 mmol) The pH was adjusted to 8.9–9.0 and kept at this value with NaOH (0.1M) until the end of the reaction (2.5–3 h, 25C) The progress of each reaction was monitored by TLC (1-butanol-2-propanol-ethylacetate-water, 2 : 4 : 1 : 3 v/v/v/v) Upon completion of the reaction, the dye was purified according to the method already described (see above) Stage 2: the purified product, VBAR-1,3-diaminopropane, was dissolved in dimethylsulf-oxide/water (3 mL, 50 : 50, v/v), introduced to a solution of shikimic acid (3 mL, 0.62 mmol), and the pH was adjusted
to 4.6, before freshly prepared solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (0.3 mL, 250 mg) was introduced dropwise under stirring over a period of 5 min, while maintaining the pH at 4.6–5.0 The reaction was stirred for a further 20 h at 25C without pH adjustment and monitored by TLC
(1-butanol-2-propanol-ethylacetate-Fig 1 Steps for the synthesis of gVBAR dye and of mimodye BM1.
Trang 5water, 2 : 4 : 1 : 3 v/v/v/v) The product,
VBAR-1,3-diami-nopropano-shikimic acid, was precipitated by addition of
solid NaCl (final content 15%, w/v), filtered and washed
with 7 mL of NaCl solution (15%, w/v) and 5 mL of cold
acetone, and dried under reduced pressure The product was
dissolved in a 50 : 50 water:methanol (50%) and
dimeth-ylsulfoxide (50%) mixture, and purified to homogeneity on
a lipophilic Sephadex LH-20 column (30· 2.5 cm) [29]
Control dyes C6NgVBAR and C3NgVBAR (Table 2) were
synthesized in the same was as in stages 1 and 2 Stages 3–5:
the purified product, VBAR-1,3-diaminopropano-shikimic
acid, was dissolved in dimethylsulfoxide/water (3 mL,
50 : 50, v/v/v) and the solution was introduced under
stirring to 1,3-diamino-2-hydroxypropane (2 mL,
0.17 mmol) The pH was adjusted to 9.0 and the mixture
was left shaking at 60C for 3 h The progress of the reaction
was monitored by TLC
(1-butanol-2-propanol-ethylacetate-water, 2 : 4 : 1 : 3 v/v/v/v) Upon completion of the
reaction, the dye was purified according to the method
already described (as above) The purified product,
1,3-
diamino-2-hydroxypropano-VBAR-1,3-diaminopropano-shikimic acid, was dissolved in dimethylsulfoxide/water
(3 mL, 50 : 50, v/v) and the pH was adjusted to 7.5 with
NaOH (0.1M) 0.2 mmol of bromoacetic acid
N-hydroxy-succinimide ester were dissolved in dioxane (1 mL) and this
solution was introduced to the dye solution The pH was
maintained at 7.5 until the end of the reaction (1.5 h, 4C,
as judged by TLC) The progress of the reaction was
monitored by TLC
(1-butanol-2-propanol-ethylacetate-water, 2 : 4 : 1 : 3 v/v/v/v) Upon completion of the
reaction, the mixture was lyophilized and the dye was
purified by applying preparative TLC as follows: lyophilized reaction mixture was dissolved in dimethylsulfoxide/water (0.4 mL, 50 : 50, v/v) and the solution applied on a Kieselgel 60 plate (silica gel 60, 0.2 mm, 20· 20 cm, Merck) The plate was developed using a 1-butanol-2-propanol-ethylacetate-water (2 : 4 : 1 : 3 v/v/v/v) mixture Following completion of the chromatography, the plate was dried and the band of interest was scraped off The desired dye was extracted from the silica gel with water, filtered through a Millipore cellulose membrane filter (0.45 lm pore size) and lyophilized The purified product, bromoacetylated 1,3-diamino-2-hydroxypropano-VBAR-1,3-diaminopropano-shikimic acid, was dissolved in 2 mL of 0.1M NaHCO3,
pH 9.0, and the solution was slowly introduced under stirring to a 2-mL solution of 0.4M 1,3-diamino-2-hydroxy-propane in 0.1MNaHCO3, pH 9.0 (the pH maintained at 9 using 1MHCl) The solution was then left under stirring for another 48–72 h (25C), without further adjustment of the
pH The progress of the reaction was monitored by TLC (1-butanol-2-propanol-ethylacetate-water, 2 : 4 : 1 : 3 v/v/ v/v) Upon completion of the reaction, the product, hydrophilic spacer-VBAR-1,3-diaminopropano-shikimic acid, was precipitated by addition of solid NaCl (final content 15%, w/v), filtered and washed with 7 mL of NaCl solution (15%, w/v) and 5 mL of cold acetone, and dried under reduced pressure The product was re-suspended in
2 mL of water and precipitated by addition of solid NaCl (final content 10%, w/v) The precipitate was filtered and washed with 7 mL each of NaCl solution (10%, w/v) and cold acetone, desiccated with 7 mL of diethyl ether and dried under reduced pressure
Table 2 The structures of the mimodyes BM1 and BM2 and the control dyes.
BM1
BM2
C6gVBAR
C6NgVBAR
C3NgVBAR
Trang 6Spectroscopic characterization and analysis
of dye-ligands
Prior to their characterization, all dyes synthesized in this
work were purified by following appropriate purification
procedures, depending on the requirements of each
syn-thetic step During the preliminary purification stage,
inorganic and certain organic contaminants were removed
by extraction with ethyl ether and precipitation with acetone In the next stage, complete dye purification was achieved on a Sephadex LH-20 lipophilic column, where salts and other organic impurities were removed Prepara-tive TLC has also been used as a purification technique at certain stages Successful dye purification was shown by TLC analysis (single blue bands) Tables 1–3 summarize the structures, molecular masses, molar absorption coefficients (e), and absorption maxima (kmax) of the purified free dyes The absorption maxima (kmax) of the purified dyes were determined by aqueous dye aliquots (50 lM) taken in the range 850–450 nm The molar absorption coefficients (e-values) were calculated from the linear section of reference curves derived by plotting dye concentration vs absorption (620 nm, 20–100 lM) [29]
NMR spectra These were recorded on a BRUKER AM
250 or 500 MHz spectrometer using standard pulse sequences Samples were analysed as solutions in dimethyl-sulfoxide-d6or D2O The ABCDand ABX patterns of the aromatics of anthraquinone and 1,4-diamino-substituted phenyl rings, respectively, are expectedly present in the1H NMR spectra of all the compounds The pattern is securely based by comparison with the1H and13C NMR spectra of the commercially purchased reference com-pound VBAR
The 1H NMR spectra of BM2 and BM1 show very complex, yet discernible high-field multiplets, ranging from
d 1.05–4.85 p.p.m and d 1.0–3.50 p.p.m., attributed to -CH-CH, -CH-NH and -CH-OH couplings, respectively Multiplets at d 5.35–6.86 p.p.m and d 5.20–6.80 p.p.m are attributed to the amide –NH resonance of both BM2 and BM1, respectively
Mass spectra Electron impact (EI) and fast atom bom-bardment (FAB) mass spectra were recorded on a VG ZAB/SE double focusing low/high resolution spectrometer Electrospray ionization (ESI) spectra were run on a Finnigan LCQ DUO spectrometer It is known that the reference compound VBAR does not exhibit a molecular ion (M+) peak under EI ionization [49] Indeed, no such ion has been observed in either EI, FAB or ESI spectra Compounds BM2 and BM1 behaved similarly However, comparing the highly complexed fragmentation patterns of BM2 and BM1, under the above ionization modes, allowed for the detection of fragments, resulting, most probably, from primary C–N and C–O fission
Immobilization of amino-dyes to carbonyldiimidazole-activated agarose and determination
of immobilized dye concentration Agarose beads (Sepharose CL-6B) were activated with 1,1¢-carbonyldiimidazole (CDI) by a modification of the pub-lished method [33] An immobilized dye concentration of approximately 2.0 lmolÆg)1Sepharose CL-6B (moist gel) was achieved by using appropriate amount of CDI in the activation step Exhaustively washed (300 mL of water) Sepharose CL-6B (600 mg, moist weight) was washed sequentially with dioxane-water (10 mL, 3 : 7, v/v), diox-ane-water (10 mL, 7 : 3, v/v), dioxane (10 mL) and dried dioxane (25 mL) The gel was re-suspended in dried dioxane (1.1 mL) to which CDI (200 mg) had already been added
Fig 2 Steps for the synthesis of mimodye BM2.
Trang 7and the mixture was tumbled for 1–2 h at 20–25 C.
Activated gel was washed with dried dioxane (20 mL) and
used immediately A solution of amino-dye (0.02 mmol) in
dimethylsulfoxide/water (2 mL, 50 : 50, v/v) was adjusted
to pH 10.0 with 2Msodium carbonate solution, whereupon
CDI activated Sepharose CL-6B (600 mg) was added The
mixture was tumbled overnight (25 C) and washed
sequentially with water (50 mL), NaCl solution (25 mL,
1M), water (25 mL), dimethylsulfoxide solution (6 mL,
50 : 50, v/v) and, finally, water (50 mL) In the case of
ligands with remaining active Cl, the adsorbent, after the
immobilization procedure, was suspended in NH3solution
(1M, pH 8.5) and tumbled for another 3 h The dyed gels
were stored as moist gels in 20% methanol at 4C Table 3
summarizes the conditions and performance of
immobil-ization reactions of the dye-ligands
Determination of immobilized dye concentration was
achieved according to [34] The concentration of the
immobilized dyes was calculated as micromoles of dye
per gram moist mass gel, using the molar absorption
coefficients shown in Table 3 All adsorbents were
substituted with dye-ligand at approximately the same
level (1.8–2.3 lmol dyeÆg moist gel)1) When comparing
affinity adsorbents, equal ligand substitution effected by
synthesis rather than dilution with unsubstituted gel is an
important but often overlooked prerequisite Wide
var-iations in immobilized ligand concentration are
undesir-able because the results obtained from the employment
of such affinity adsorbents may lead to misleading
conclusions Extreme levels of ligand substitution may
lead to no binding, due to the steric effect caused by the
large number of dye molecules, or even to nonspecific
protein binding [47,48]
Assay of enzyme activities and protein, and inactivation
of galactose dehydrogenase by VBAR
Galactose dehydrogenase (GaDH), galactose oxidase
(GaO), glucose oxidase (GlO), glucose dehydrogenase
(GlDH) and alcohol dehydrogenase (ADH) assays were
performed at 25C with the exception of GlO, which was performed at 35C The assays were performed according
to [35] [36], [37], [38], and [39], respectively All assays were performed in a double beam UV-visible spectrophotometer equipped with a thermostated cell holder (10-mm path-length) For GaO, one unit of enzyme activity is defined as the amount that produces a DA425nmof 1.0 per min at the conditions of the assay For the rest of the enzymes, one unit
of enzyme activity is defined as the amount that catalyses the conversion of 1 lmol of substrate to product per min Protein concentration was determined by the method of Bradford [40] or by a modified Bradford’s method [41], using bovine serum albumin (fraction V) as standard Inactivation of GaDH by VBAR was performed in incubation mixture containing in 1 mL total volume (35C): 100 lmol Hepes/NaOH buffer pH 8.5, 30 nmol VBAR, 0.13 U GaDH (enzyme assay at 25C) The rate of GaDH inactivation was followed by periodically removing samples (100 lL) from the incubation mixture for assay of enzymatic activity Competitive inactivation studies of GaDH by VBAR were performed in the above reaction mixture of 1 mL total volume (35C) containing also
1 lmol NAD+ Preparation of cell extracts with enzyme activities
P fluorescensdry cells (1.5 mg) were suspended in 1 mL of
10 mM potassium phosphate buffer containing 1 mM
EDTA, pH 6.5, 7.0 or 7.5, and ultrasonically disintegrated (Vibra Cell, 400 Watt, Sonics & Materials) (amplitude: 40%, 2 s sonication, 5 s pause, 8 cycles, 4C) Cell debris was removed by centrifugation (5000 g, 20 min, 4C) and the supernatant was dialyzed overnight at 4C against 2 L
of 10 mM potassium phosphate buffer containing 1 mM
EDTA, pH 6.5, 7.0 or 7.5 The dialysate was clarified through a Milipore cellulose membrane filter (0.45 lm pore size), affording, typically, 0.08 U GlDHÆmL)1 extract (0.05 U GlDHÆmg dry cells)1) In the case of GaD H, before dialysis, the supernatant was enriched as necessary with commercial enzyme (P fluorescens gene expressed in
Table 3 Characteristics of free biomimetic and nonbiomimetic dyes,and conditions and performance of their immobilization reactions onto agarose.
Dye-ligand
M r
(sodium salt)
me (m M )1 Æcm)1)
in water
k max (nm)
in water
mg dye per g moist gel (in reaction)
lmol dye per g moist gel (in adsorbent)
me a
(m M )1 Æcm)1)
a
Determined in medium identical to the one that resulted after acid hydrolysis of the adsorbent Values were determined from 20 l M dye solutions made in the above medium The duration of all reactions was 18 h.
Trang 8E coli) in order to achieve an initial specific activity of
about 1.1 U GaDHÆmg)1
Commercial lyophilized crude powder (10 mg) of
Dacty-lium dendroideswas suspended in 2 mL of 100 mM
potas-sium phosphate buffer, pH 7.0 or 7.5 and the suspension
was centrifuged (5000 g, 20 min, 4C) The supernatant
was dialyzed overnight at 4C against 2 L of 100 mM
potassium phosphate buffer, pH 7.0 or 7.5 The dialysate
was clarified through a Millipore cellulose membrane filter
(0.45 lm pore size), affording specific activity, typically,
of 51.3 U GaOÆmg)1 (18.3 U GaOÆmL)1 extract, 3.7 U
GaOÆmg cell lyophilized powder)1)
Commercial lyophilized crude powder (11 mg) of
Aspergillus niger was suspended in 1.5 mL of 10 mM
potassium phosphate buffer containing 1 mM EDTA,
pH 7.0 or 7.5 The suspension was centrifuged (5000 g,
20 min, 4C) and the supernatant was dialyzed
over-night at 4C against 2 L of 10 mM potassium
phosphate buffer containing 1 mM EDTA, pH 7.0 or
7.5 The dialysate was clarified through a Millipore
cellulose membrane filter (0.45 lm pore size), affording,
typically, 11.2 U GlOÆmL extract)1 (1.5 U GlOÆmg
solid)1)
Commercial baker’s yeast cells (9 g paste) were
suspen-ded in 12 mL of 10 mM potassium phosphate buffer
containing 1 mMEDTA, pH 7.0 or 7.5, or 10 mM
potas-sium phosphate buffer, pH 6.5, 7.0 or 7.5, before
ultra-sonically disintegrated (amplitude 40%, 5 s sonication, 5 s
pause, 12 cycles, 4C) Cell debris was removed by
centrifugation (14 000 g, 50 min, 4C) and the supernatant
was dialyzed overnight at 4C against 2 L of 10 mM
potassium phosphate buffer containing 1 mM EDTA,
pH 7.0 or 7.5, or 10 mM potassium phosphate buffer,
pH 6.5, 7.0 or 7.5 The dialysate was clarified through a
Milipore cellulose membrane filter (0.45 lm pore size),
affording, typically, an activity of 0.06 U GaDHÆmL
extract)1(0.08 U GaDHÆg cell paste)1), 0.39 U GlDHÆmL
extract)1 (0.52 U GlDHÆg cell paste)1) and 5.5 U
ADHÆmL extract)1(7.3 U ADHÆg cell paste)1)
Green peas (13 g) were suspended in 20 mL of 10 mM
potassium phosphate buffer containing 1 mM EDTA, 7.0
or 7.5, or 10 mMpotassium phosphate buffer, pH 6.5, 7.0
or 7.5, before pulped using pestle and mortar, and
homogenized (Virtishear mechanical homogenizer,
10 000 r.p.m., 1 min, 4C) The homogenized suspension
was filtered using cheese cloth and the filtrate was
centrifuged (18 000 g, 40 min, 4C) The supernatant
was dialyzed overnight at 4C against 5 L of 10 mM
potassium phosphate buffer containing 1 mM EDTA,
pH 7.0 or 7.5, or 10 mM potassium phosphate buffer,
pH 6.5, 7.0 or 7.5 The dialysate was clarified through a
Milipore cellulose membrane filter (0.45 lm pore size),
affording, typically, an activity of 0.02 U GaDHÆmL
extract)1 (0.03 U GaDHÆg)1) and 0.3 U AD HÆmL
ex-tract)1(0.46 U ADHÆg)1)
Rabbit liver (5 g) was suspended in 20 mL of 10 mM
potassium phosphate buffer containing 1 mM EDTA,
pH 6.5 or 7.0, and homogenized (Virtishear mechanical
homogenizer, 10 000 r.p.m., 3 min, 4C) The
homogen-ized suspension was centrifuged (750 g for 15 min, 4C)
and the supernatant was re-centrifuged (14 000 g, 50 min,
4C) The supernatant was dialyzed overnight at 4 C
against 5 L of 10 m potassium phosphate buffer, pH 6.5
or 7.0 The dialysate was clarified through a Milipore cellulose membrane filter (0.45 lm pore size), affording, typically, an activity of 0.03 U GaDHÆmL extract)1 (0.12 U GaDHÆg)1)
Affinity chromatography evaluation of the amino-alkyl-dyes, hydrophilic spacer-dye and control-dyes using GaDH fromP fluorescens extract
All procedures were performed at 4C Galactose dehy-drogenase binding was assessed using analytical columns, each packed with 0.5 mL of adsorbent bearing immobilized ligand (amino-alkyl-VBAR dyes, structures aVBAR-fVBAR and hydrophilic spacer-VBAR dye, structure gVBAR of Table 1, as well as control-dyes, structures
C6gVBAR, C6NgVBAR and C3NgVBAR of Table 2) (1.8– 2.3 lmol dyeÆg moist gel)1) Columns were equilibrated with 10 mMpotassium phosphate buffer containing 1 mM
EDTA, pH 7.0 Dialyzed P fluorescens extract (0.5– 1.0 mL, 0.1–0.2 U GaDH, 0.09–0.17 mg protein) was applied to each analytical column Non-adsorbed protein was washed off with equilibration buffer (2–3 mL) Bound GaDH activity was eluted with 2 mL equilibration buffer containing a mixture of 1 mMNAD+and 10 mMNa2SO3 Collected fractions (1 mL) were assayed for GaDH activity
Affinity chromatography evaluation of mimodye adsorbents using GaDH fromP fluorescens extract
All procedures were performed at 4C Galactose dehy-drogenase binding was assessed using analytical columns, each packed with 0.5 mL of mimodye adsorbent (1.8–2.2 lmol dyeÆg moist gel)1) Columns were equili-brated with 10 mM potassium phosphate buffer at the pHs shown in Table 4, containing 1 mMEDTA Dialyzed
P fluorescens enriched extract (0.5–1.0 mL, 0.10–0.38 U GaDH, 0.09–0.33 mg protein) was applied to each analyt-ical column Non-adsorbed protein was washed off with equilibration buffer (2–4 mL) Bound GaDH was eluted, from immobilized BM1, by a mixture of 0.5 mMNAD+ and 5 mMNa2SO3in the equilibration buffer (2–4 mL) or, from immobilized BM2, by 0.8 mM NAD+ and 8 mM
Na2SO3 in the equilibration buffer (3–4 mL) Collected fractions (1 mL) were assayed for GaDH activity and protein [41] The fractions with GaDH activity were pooled and the specific activity was determined
Table 4 Affinity chromatography evaluation of immobilized mimodyes and hydrophilic spacer-VBAR dye for binding GaDH activity from
P fluorescens crude extract.
Dye-ligand pH
SA (unitsÆmg)1)
Purification (-fold)
Recovery (%) BM1 6.5 29.9 27.2 66
7.0 48.2 41.9 100 7.5 30.3 27.5 35 BM2 6.5 15.1 13.7 68
7.0 37.7 32.8 76 7.5 41.2 37.5 98 8.0 28.8 25.3 28 gVBAR 7.0 15.4 13.4 33
Trang 9Affinity chromatography control experiments for the
evaluation of the binding selectivity of mimodye
adsorbents using enzymes other than GaDH
On each of the mimodye adsorbents (0.5 mL), previously
equilibrated with 10 mM potassium phosphate buffer
(pH 7.0 for BM1 and 7.5 for BM2) containing 1 mM
EDTA, were applied the enzyme units shown in Table 5,
previously dialyzed in the same equilibration buffer (4C)
After the column was washed with equilibration buffer,
elution of bound proteins was effected with 1MKCl in the
same buffer In the case of GaO, the equilibration buffer
used was 100 mMpotassium phosphate (pH 7.0 for BM1,
pH 7.5 for BM2)
R E S U L T S A N D D I S C U S S I O N
Protein molecular modelling
Fold recognition results were near-unanimous in
highlight-ing the structure of Zymomonas mobilis glucose-fructose
oxidoreductase (GFO; PDB code 1ofg;) as the best available
template for GaDH model construction For example, the
3D-PSSM method [16] gave GFO a score of 6· 10)6with the next best hit scoring 95· 10)3 Similarly, the FFAS method [17] gave GFO a score of 67 and the next best template just 14 In each case, these results are strongly significant for GFO and show it to be much more suitable as GaDH template than the next best structures The only exception to the trend wasGENTHREADER[15] which gave GFO and rat biliverdin reductase similar high probabilites
of 0.94 and 0.95, respectively In fact GFO, rat biliverdin reductase and many others of the better scoring hits of the fold recognition studies, all catalyse redox reactions and are structurally related, sharing a dinucleotide binding fold, in conjunction with a more variable domain responsible principally for substrate binding [42]
Based on the FFAS alignment an initial GaDH-GFO target-template alignment was constructed by examination
of the GFO structure to determine the most likely positions
at which the 10 insertions or deletions could be accommo-dated In most cases these positions were between secondary structure elements but in others, the size of the insertion or deletion naturally led to alteration of neighbouring helices
or strands (see Fig 3) Although GaDH is a dimer, the regions of the alternate subunit corresponding to those that
Table 5 Control experiments for the evaluation of the binding selectivity of immobilized BM1 and BM2 with enzymes other than GaDH On each affinity adsorbent (0.5 mL), previously equilibrated with 10 m M potassium phosphate buffer containing 1 m M EDTA (pH 7.0 for BM1, pH 7.5 for BM2), were applied the enzyme units shown, previously dialyzed in the same equilibration buffer as above (4 C) After the adsorbent was washed with equilibration buffer, elution of bound proteins was effected with 1 M KCl For GaO, 100 m M potassium phosphate buffer (pH 7.0 for BM1,
pH 7.5 for BM2) was used as the equilibration buffer.
Enzyme Source Units applied Bound enzyme (%) Units applied Bound enzyme (%) GaO Dactylium dendroides 15.7 5.8 13.8 5.2
GlO Aspergillus niger 8.3 6.2 4.6 12.6
Fig 3 ALSCRIPT [52] alignment of GaDH with template GFO The secondary structure of GFO is shown above the alignment and residues shared between the two proteins are emboldened.
Trang 10in GFO contribute to cofactor and substrate binding (the
N-terminal stretch and the loop around GFO residue 317),
are not present in GaDH (Fig 3) Therefore, modelling of
an individual monomer was undertaken The first set of 20
models was, thus, constructed and analysed as outlined in
Materials and methods At regions of improbable protein
packing and solvent exposure, as indicated byPROSAII, a
series of alignment variants was constructed These variants,
typically, involved 1–3 residues shifts of single secondary
structure elements, with the flanking loops accommodating
correspondingly altering in length These were analysed and
the process repeated until no further alignment
improve-ments could be found In all 17 different alignimprove-ments were
tested Special attention was then paid to stereochemical
aspects of the model Residues in disallowed or generously
allowed areas of the Ramachandran plot were treated as
possible errors and dealt with either by flipping of peptide
bonds or ab initio regeneration withMODELLER At the end
of this process the structure best combining lowPROSAII
score and good stereochemistry was taken as the final
model
As previously observed, significant improvements in
model quality resulted from this careful construction
procedure The first set of models hadPROSAII scores in
the range)7.8 to )8.7 For the final model this improved to
)10.1 Comparison with the template also suggests a model
of high objective quality The somewhat longer GFO (351
residues vs 303 in the final model) scores)11.5 byPROSAII
analysis The overall stereochemical quality of the model
and GFO, as measured by the G-factor calculated by
PROCHECK, is near-identical;)0.15 for the model, )0.16 for
the crystal structure The GaDH model places 90.5% of
residues in most-favoured regions of the Ramachandran
plot, suggestive of good structural quality and similar to the
91.5% value of the GFO template As well as these overall
indicators, it is worth remembering that the isolated regions
of high sequence identity between target and template,
around GaDH positions 10 and 85 (Fig 3), are situated
near the cofactor binding site Hence, this part of the final
structure, important for docking studies, should be
parti-cularly well-modelled
Ligand design and docking
With the good objective quality of the GaDH model
established, docking experiments were initiated to indicate
possible galactosyl-biomimetic ligands for GaDH The
three ring systems of the CB3GA-derived portion
(num-bered 1–3: anthraquinone, diaminobenzosulfonic acid and
triazine, respectively; see Fig 4A and Table 1, VBAR) were
first docked into the GaDH model, followed sequentially by
the galactose portion, the linker molecule between ring 3
and the galactose, and finally the chain (spacer molecule)
by which the ligand attaches to the chromatograpic matrix
(e.g agarose beads) Experimental evidence regarding
residues involved in substrate and cofactor binding to
GaDH is entirely lacking, and inference of possible
important regions through their sequence conservation is
rendered impossible by the lack of any known close GaDH
homologues Nevertheless, a variety of other indirect data
could be used to guide the docking
The knowledge that ring systems 1–3 bind in NAD(P)
binding sites, with the anthraquinone ring system 1
Fig 4 Stereo MOLSCRIPT [53] diagrams showing interactions of unre-fined,docked components with the final GaDH model (A) Ring systems 1–3 (B) galactose (presumed to bind similarly to 2-amino-2-deoxyga-lactose) and (C) shikimic acid Hydrogen bonds are shown by dotted lines.