(Received for publication, July 22, 1996, and in revised form, November 19, 1996)
From the Institute of Pathology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106
Four "amadoriase" enzyme fractions, which
oxidatively degrade glycated low molecular weight amines and amino
acids under formation of hydrogen peroxide and glucosone, were isolated
from an Aspergillus sp. soil strain selected on fructosyl
adamantanamine as sole carbon source. The enzymes were purified to
homogeneity using a combination of ion exchange, hydroxyapatite, gel
filtration, and Mono Q column chromatography. Molecular masses of
amadoriase enzymes Ia, Ib, and Ic were 51 kDa, and 49 kDa for
amadoriase II. Apparent kinetic constants for
N-fructosyl
N
-t-butoxycarbonyl lysine
and fructosyl adamantanamine were almost identical for enzymes Ia, Ib,
and Ic, but corresponding values for enzyme II were significantly
different. FAD was identified in all enzymes based on its typical
absorption spectrum. N-terminal sequence was identical for enzymes Ia
and Ib
(Ala-Pro-Ser-Ile-Leu-Ser-Thr-Glu-Ser-Ser-Ile-Ile-Val-Ile-Gly-Ala-Gly-Thr-Trp-Gly-) and Ic except that the first 5 amino acids were truncated. The sequence of enzyme II was different
(Ala-Val-Thr-Lys-Ser-Ser-Ser-Leu-Leu-Ile-Val-Gly-Ala-Gly-Thr-Trp-Gly-Thr-Ser-Thr-). All enzymes had the FAD cofactor-binding consensus sequence
Gly-X-Gly-X-X-Gly within the
N-terminal sequence. In summary, these data show the presence of two
distinct amadoriase enzymes in the Aspergillus sp. soil
strain selected on fructosyl adamantanamine and induced by fructosyl
propylamine. In contrast to previous described enzymes, these novel
amadoriase enzymes can deglycate both glycated amines and amino
acids.
Nonenzymatic glycation of proteins has been implicated in the
pathogenesis of diabetic complications as one of several mechanisms by
which chronic elevation of glycemia may be responsible for the early
cellular and extracellular dysfunctions triggered by diabetes. The
Amadori product, which forms in the initial stages of the Maillard
reaction in vivo, has been shown to be recognized by
receptors at the surface of monocytes, aortic endothelial, and
mesangial cells (1-3), and treatment of diabetic mice with antibodies
to glycated albumin decreases the rate of progression of diabetic
nephropathy (4). Amadori products of glucose are precursors of the
glycoxidation products pentosidine and
N-(carboxymethyl)lysine
(5-7), which are elevated in diabetes and the levels of which increase
with the severity of diabetic complications (8-10). Furthermore,
N
-(carboxymethyl)lysine has
now been identified as the major advanced glycation end product
(AGE)1 epitope (11) and is also likely to
be responsible for the major negative charge in AGE proteins which
appears to play a role for recognition by AGE receptors in macrophages
or endothelial cells (12, 13). Amadori products and glycated
proteins are able to generate free radicals, which may oxidize low
density lipoproteins and form covalent cross-links with other proteins
(14, 15).
Collectively, these data suggest that increased levels of Amadori
product in diabetes may be responsible for much of the glucotoxicity underlying the pathogenesis of diabetic complications. Based on this
premise, we began a few years ago to search for novel ways to
selectively prevent the effects of protein glycation by attempting to
deglycate proteins enzymatically (16), with the ultimate goal of
utilizing such enzymes in transgenic models of hyperglycemia. We found
and partially purified a deglycating enzyme in a Pseudomonas sp. soil strain selected for growth with glycated -aminocaproic acid
as sole carbon source (17). Surprisingly, however, the enzyme obtained
cleaved glycated substrates at the N-alkyl instead of the
ketoamine bond and released free fructosamine, while the deaminated
alkyl residue spontaneously oxidized to the acid.
We synthesized a highly sterically inhibited Amadori product using adamantanamine, and utilized this substrate as sole carbon source to select soil organisms. Two molecularly distinct isoenzymes were obtained from an Aspergillus sp., which can degrade both glycated low molecular weight amines and amino acids. The work described below provides a basis for the molecular analysis of deglycating enzymes and their biological mechanism of regulation.
Materials
N,O-Bis(trimethylsilyl)acetamide (BtmSA) was obtained
from Fluka. Silica gel-coated aluminum thin layer chromatography plates (0.2 mm thick) were obtained from EM Separations (Gibbstown, NJ). -Glucosone was a gift from Dr. Milton Feather (Department of Biochemistry, University of Missouri) or synthesized from the phenylosazone derivative (18) as described by Bayne (19). Adamantanamine and o-phenylenediamine (OPD) were
purchased from Aldrich. Aminopropyl glass, poly-D-lysine,
poly-L-lysine, phenylmethylsulfonyl fluoride (PMSF), EDTA,
bovine serum albumin (BSA) (fraction V) and
m-aminophenylboronic acid agarose were from
Sigma. Glucosone triazine was a gift from Dr. Marcus
Glomb. DEAE-Sepharose, Sephacryl S-200, and Mono Q were purchased from
Pharmacia (Uppsala, Sweden). Bio-Gel HT hydroxyapatite was obtained
from Bio-Rad. All other materials were analytical grade.
Synthesis of Glycated Substrates
Fructosyl adamantanamine was synthesized according to a modified
protocol of Hodge and Rist (20). 5 g of adamantanamine, 3.8 g
of glucose, and 10 ml of acetylacetone were heated in 100 ml of ethanol
for 2 h under reflux. The solution was evaporated, and the residue
was purified by silica gel column chromatography (17 × 2.5 cm,
eluent: n-butanol/NH3, 10:1; monitored by TLC,
eluent: ethanol/NH3, 10:1, RF = 0.4, detected with 0.2% ninhydrin in ethanol or 2% TTC in methanol, 1 N NaOH, 1:1). Fractions containing the product were pooled,
evaporated, redissolved in water, and freeze-dried. The product
identity was confirmed by 1H NMR: 1.36-1.70 (12H), 2.05 (3H), 3.31 (2H), and 3.52-3.56 (5H). Glycated BSA,
poly-D-lysine (Mr 150,000-300,000),
and poly-L-lysine (Mr
30,000-70,000) were prepared as follows: 15 mg/ml BSA,
poly-D-lysine or poly-L-lysine was incubated in
50 mM HEPES (pH 7.4) containing 1 M glucose, 1 mM DTPA, and 1 µl/ml chloroform and toluene for 5 days at
37 °C. The pH was monitored during incubation and adjusted by NaOH.
The solution was dialyzed three times against water and freeze-dried.
Glycated aminopropyl glass (Sigma) and
Spherisorb-NH2 (Phenomenex) were prepared as follows; 50 mg/ml aminopropyl glass or Spherisorb was incubated in
phosphate-buffered saline (PBS; 137 mM NaCl, 2.68 mM KCl, 1.47 mM KH2PO4,
8.06 mM Na2HPO4) containing 1 M glucose, 1 mM DTPA, and 1 µl/ml chloroform
and toluene for 24 h at 60 °C. The suspension was filtered and
intensively washed with sterile water.
N-fructosyl
N
-t-Boc-lysine and
N
-fructosyl
N
-acetyllysine were prepared according
to Finot and Mauron (21). Fructosyl propylamine was prepared as
follows; 30 g of glucose and 30 ml of propylamine were heated at
70 °C until the sugar was dissolved. The mixture was diluted in 200 ml of isopropanol and poured into 800 ml of diethyl ether. The
precipitate was filtered and dissolved in 300 ml of boiling dioxane.
20 g of oxalic acid dissolved in 200 ml of dioxane was added, and
the mixture was heated for 15 min at 70 °C. The precipitate was
crystallized from dioxane/methanol (1:1), and oxalate was replaced by
chloride by anion exchange chromatography. Fructosyl propylamine in
reduced form was prepared as follows; 1.2 g of NaBH4
was added to 200 mg of fructosyl propylamine dissolved in 50 ml of
water and incubated 12 h at room temperature. 1 ml of acetic acid
was added to stop the reaction, and the solvent was removed under
reduced pressure. 50 ml of methanol was added and evaporate to remove
boronic acid. The residue was purified by Sephadex G-15 gel filtration
column chromatography (eluent: PBS, detected with 0.2% ninhydrin in
ethanol).
Microorganism and Culture Media
An Aspergillus sp. strain was isolated from soil by selection on minimal medium (3 g of KH2PO4, 7 g of K2HPO4, 0.1 g of (NH4)2SO4, 1 mM MgSO4 in 100 ml), containing fructosyl adamantanamine as sole carbon source (5.5 mg/ml). Solid media containing 2 g of agar/100 ml of medium were also prepared. The microorganism was isolated by alternate growth in liquid and on solid medium. Batch cultures were grown in nutrient broth containing 1 mg/ml fructosyl propylamine. Incubation was carried out at 37 °C for 3 days aerobically.
Preparation of an Enzyme-rich Fraction
To identify the reaction products, enzyme-rich fraction was prepared from Aspergillus sp. as follows. Mycel was harvested by filtration and washed by PBS. 750 mg (wet weight) of mycel was homogenized by using Potter-Elvehjem Teflon-glass homogenizer and by vortexing with 900 mg of glass beads in 300 µl of 20 mM sodium phosphate buffer, pH 7.4, containing 1 mM PMSF, 1 mM EDTA, and 2 mM DTT. The homogenate was centrifuged for 1 h at 100,000 × g, and the supernatant was brought to ammonium sulfate fractionation. The precipitation between 45% and 70% of ammonium sulfate was obtained by centrifugation at 15,000 × g for 15 min, redissolved in PBS containing 1 mM EDTA and 0.2 mM PMSF and diluted to a protein concentration of 8.0 mg/ml.
Identification of Enzymatic Degradation Products
The reaction products were identified by TLC and GC/MS. As
a control an enzyme solution was heated for 15 min at 100 °C. For TLC 200 µl of the enzyme-rich fraction and 2 mg of
N-fructosyl
N
-t-Boc-lysine or fructosyl
propylamine were incubated at 37 °C. After 1-4 days a sample was
tested on TLC plates (eluent: n-butanol/acetic acid/water,
5:3:1, detection: 0.2% ninhydrin in ethanol or 2% TTC in methanol, 1 N NaOH, 1:1). As standards
N
-fructosyl
Na-t-Boc-lysine, fructosyl
propylamine, glucosone, glucose, mannose, N
-t-Boc-lysine, and
propylamine were used. For GC/MS 8 µl of enzyme-rich fraction was
incubated in 1 ml of PBS containing 15 mM fructosyl propylamine and 50 mM aminoguanidine for 24 h at
37 °C. The solution was filtered through a Microcon-10 (Amicon), and
the filtrate was dried under vacuum (Savant, Farmingdale, NY). The
residue was derivatized in 100 µl of pyridine/BtmSA (1:1) for 1 h at room temperature. Coupled gas chromatography and mass spectroscopy (GC/MS) were performed on a Hewlett-Packard 5890 series II gas chromatograph using helium as the carrier gas (flow rate 26.3 cm/s) on
an Ultra 2 capillary column (25 m, 0.2 mm (inner diamter), film
thickness 0.33 µm; Hewlett-Packard). Methane was used for positive
chemical ionization. Injector and interface temperatures were 270 °C
and 280 °C, respectively. Temperature program was 100-200 °C at
5 °C · min
1, 200-270 °C at 10 °C · min
1, and isothermal at 270 °C for 10 min. Glucosone
triazine generously provided by Dr. Marcus Glomb was derivatized as
above and used as a standard.
Assays for Enzymatic Activity
Four different assays were developed to measure the activity of the enzyme.
Glucosone FormationFor purification, the enzyme activity was monitored by the release of glucosone measured by a colorimetric reaction with OPD (22) using fructosyl propylamine as a substrate. This assay is based on the end point measurement of glucosone formed after 120 min of reaction time. The reaction mixture contained 20 mM sodium phosphate, pH 7.4, 10 mM OPD, 10 mM fructosyl propylamine, and enzyme protein in a final volume of 1 ml. After incubation at 37 °C for 2 h, the absorbance at 320 nm was measured. The reaction was linear to 240 min in a dose-dependent manner under these conditions. One unit of enzyme activity was defined as the amount of the enzyme that produces 1 µmol of glucosone/min. Synthesized glucosone was used as a standard.
Free Amine AssayTo assay the release of free amine,
fluorescence was measured after reaction with fluorescamine. 25 µl of
a solution of pure enzyme or enzyme-rich fraction, 15 µl of 20%
fructosyl propylamine in water, and 250 µl of PBS were incubated at
37 °C for different times as indicated. The reaction was stopped by
filtration through a Microcon-10 (Amicon, Beverly, MA) at 4 °C. 1 µl of the pure or 1:10 diluted filtrate was added to 1.5 ml of 50 mM phosphate buffer pH 8.0. Under vigorous vortexing
0.5 ml of 0.03% fluorescamine in dioxane was rapidly added. After 5 min fluorescence was measured (ex = 390 nm,
em = 475 nm). A standard plot was made with 6-150 ng of
propylamine.
Hydrogen peroxide was
quantitated by the quinone dye assay according to Sakai et
al. (23). The reaction mixture contained 20 mM
Tris-HCl, pH 8.0, 1.5 mM 4-aminoantipyrine, 2.0 mM phenol, 2.0 units of peroxidase, 10 mM
fructosyl propylamine, and enzyme protein in a total volume of 1 ml.
Production of the H2O2 was monitored by the
formation of a quinone dye following the absorbance at 505 nm ( = 5.13 × 103). The production of 0.5 µmol of quinone
dye corresponds to the formation of 1.0 µmol of
H2O2.
Oxygen consumption was determined with a YSI-Beckman glucometer II equipped with a Clarke type oxygen electrode as described before (17). Briefly, enzyme (50 µl) was added to the chamber containing 750 µl of PBS and 650 µl of water. The reaction was started by addition of 50 µl of 300 mM fructosyl propylamine (final concentration 10 mM).
Purification of the Enzyme
All purification steps were carried out at 4 °C. Washed mycelia (180 g as wet weight) were suspended and homogenized in 900 ml of homogenizing buffer (20 mM sodium phosphate buffer, pH 7.4, 1 mM PMSF, 1 mM EDTA, 2 mM DTT) using a Polytron homogenizer (Kinematica, Littau, Switzerland) and Potter-Elvehjem Teflon-glass homogenizer. The homogenate was squeezed through the gauze and centrifuged at 20,000 × g for 30 min to remove unbroken cell and cell debris. The pellet was discarded, and solid ammonium sulfate was added to the supernatant to give 45% saturation. After stirring for 30 min, the mixture was centrifuged at 6,000 × g for 30 min. The supernatant was adjusted to 70% saturation by solid ammonium sulfate. After stirring for 30 min, the precipitate was obtained by centrifugation at 6,000 × g for 30 min and dissolved in a minimum volume of buffer A (20 mM Tris-HCl buffer, pH 8.0, 1 mM EDTA, 2 mM DTT) followed by dialysis against the same buffer. Insoluble material formed was removed by centrifugation at 20,000 × g for 15 min. The supernatant was applied to a DEAE Sepharose column (5 × 16 cm) equilibrated with buffer A. The column was washed extensively with buffer A, and the bound protein was eluted with a 3000-ml linear gradient of NaCl of 0-0.2 M in the same buffer. Fractions containing enzyme activity were pooled and dialyzed against 10 mM sodium phosphate buffer, pH 7.4, containing 1 mM EDTA, 2 mM DTT and applied to a hydroxyapatite column (1.5 × 4 cm) equilibrated with the same buffer. After washing the column extensively with the equilibration buffer, elution was carried out with a 140-ml linear gradient of sodium phosphate buffer, pH 7.4, over 10-100 mM, containing 1 mM EDTA and 2 mM DTT. Fractions with enzyme activity were pooled and concentrated with an Amicon Diaflow Ultrafiltration using PM 10 membrane (Amicon), and applied to a Sephacryl S-200 column (1 × 90 cm) equilibrated with 20 mM sodium phosphate buffer, pH 7.4, containing 150 mM NaCl, 1 mM EDTA, and 2 mM DTT, and eluted with the same buffer. The enzyme-active fractions were pooled, dialyzed against buffer A, and applied onto a Mono Q column (0.5 × 5 cm) equilibrated with buffer A. The column was washed with 4 ml of buffer A, and elution was carried out with a 20-ml linear gradient of 0-0.5 M NaCl in buffer A. Fractions containing enzyme activity were used as the purified preparation of amadoriases.
Protein Determination
Protein concentration was determined by the method of Bradford (24) with a Bio-Rad protein assay kit using BSA as a standard. Protein analysis by SDS-PAGE was carried out according to Laemmli (25). The molecular weight of native enzyme was determined by gel filtration on a Sephacryl S-200 column equilibrated with PBS containing 2 mM DTT using molecular weight markers (Sigma).
Kinetic Constants
Kinetic constants were calculated from the data by least-squares linear regression analysis.
Determination of pH Profiles and Isoelectric Points
pH profiles were determined over the range pH 5.8-9.5 using the following buffers: MES (pH 5.8-6.5), MOPSO (pH 6.5-7.5), POPSO (pH 7.5-8.5), and CHES (pH 8.5-9.5). Overlaps were used in all cases, and checks were made to ensure that none of the buffers were inhibitory and that the enzymes was stable during the time needed for the measurements. Isoelectric points were determined by isoelectric focusing using PhastGel IEF 5-8 (Pharmacia) and pI maker (IEF Mix 3.6-9.3, Sigma) in PhastSystem (Pharmacia).
Amino Acid Sequencing
N-terminal amino acid sequencing was performed at the Molecular Biology Core laboratory (Department of Biochemistry, Case Western Reserve University) with an Applied Biosystems 477A Protein Sequencer (Foster City, CA).
Soil specimens were screened for a microorganism that can degrade Amadori products by using culture media with glycated substrates as sole carbon source. In order to increase the chances that the enzyme would also have activity against glycated proteins, Amadori products were chosen with a maximal steric hindrance at the amino side. As carbon sources glycated aminopropyl glass, glycated aminopropyl silica gel, glycated poly-D-lysine, and fructosyl adamantanamine were tested, but no organism could be isolated in presence of these substrates except fructosyl adamantanamine.
From the medium enriched with 5.5 mg/ml fructosyl adamantanamine, a
microorganism was isolated and identified as an Aspergillus sp. strain. To ascertain the presence of Amadori product degrading activity, an extract of the fungus was produced and incubated with
N-fructosyl
N
-t-Boc-lysine. The
reaction was monitored by TLC (Fig. 1) and compared with
that of the heat-inactivated extract. In the course of the incubation
the amount of the Amadori product in the extract was decreasing,
whereas two new spots appeared on TLC. A ninhydrin-positive spot could
be identified by comparison of the RF with an
authentic standard as free
N
-t-Boc-lysine. The other
spot reacted with TTC and co-migrated with synthetic glucosone. In the
same way, free propylamine and glucosone derived from the reaction
mixture of enzyme-rich fraction and fructosyl propylamine were observed
(data not shown).
Identification of the Reaction Products
In order to confirm
the molecular identity of the products, GC/MS analysis was performed
after preparing the triazine derivatives with aminoguanidine and
the trimethylsilyl derivatives with BtmSA. In the sample two major
peaks appeared at retention times of 26.13 and 26.60 min, respectively
(Fig. 2, top panel). The mass spectrum of
both compounds showed characteristic peaks at m/z 342, 270, and 217 (Fig. 2A). A sample of authentic glucosone was
derivatized in the same way with aminoguanidine and BtmSA and subjected
to GC/MS analysis. The chromatogram showed peaks at 26.13 and 26.60 min
for the two triazine stereomers, and the fragmentation patterns were
identical to those of the degradation products (Fig. 2B). From the TLC and GC/MS data, it can be concluded that the enzyme releases glucosone and free amine from the Amadori product. Oxygen consumption during the reaction was detected polarographically using an
oxygen electrode, and H2O2 generation was
detected by the quinone dye assay (data not shown). Thus, all the data
confirm that the Amadori product is degraded by an oxidative
cleavage of the ketoamine bond, whereby oxygen is used as an electron
acceptor and free amine and glucosone are released (Fig.
3).
Enzyme Assays and Time and Dose Dependence
Time-course
experiments on the formation of glucosone detected as quinoxaline
derivative by the OPD method were carried out with an enzyme-rich
fraction incubated with fructosyl propylamine (Fig. 4).
The amount of glucosone formed increased in the course of the
incubation and with increasing enzyme concentration, proving thereby that an enzyme was responsible for the activity. A
time-dependent release of free amine could be also found by
free amine assay using fluorescamine (data not shown).
Purification and Characterization of Four Enzyme Fractions
Since glucosone could be easily assayed by the OPD
method, it was used for routine assays in the course of purification.
Purification was performed as described under
"Experimental Procedures." The extract was first
fractionated by DEAE-Sepharose (Fig. 5A). Two peaks of amadoriase activity were observed. They were pooled into "pool 1" and "pool 2" and separately subjected to further
purification. Fig. 5B shows the profile of the
hydroxyapatite column chromatography of pool 1. Three peaks of activity
were observed and purified individually by gel filtration and Mono Q
column chromatography. Obtained enzymes were named amadoriase Ia, Ib,
and II according to the order of elution from the hydroxyapatite column
chromatography. Pool 2 was also subjected to hydroxyapatite column
chromatography (Fig. 5C), and an active peak obtained was
purified by gel filtration and Mono Q column chromatography. The enzyme
obtained from pool 2 was named amadoriase Ic. Altogether four
amadoriase enzyme fractions were isolated from Aspergillus
sp. that had been selected with fructosyl adamantanamine and grown in
the presence of fructosyl propylamine. The results of each purification
of amadoriases are summarized in Table I. Each enzyme
was purified to homogeneity as judged by Coomassie-stained SDS-PAGE
(Fig. 6). The apparent molecular mass of amadoriase Ia,
Ib, and Ic were found to be 51 kDa, and that of amadoriase II was 49 kDa. From the results of Sephacryl S-200 gel filtration, the apparent
molecular masses of native amadoriases Ia, Ib, and Ic were
calculated to be 40 kDa and II was 55 kDa (data not shown). The results
indicated that all four enzymes were found to be monomers.
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Substrate Specificity and Kinetic Constants of the Purified Amadoriases Ia, Ib, Ic, and II
Substrate specificity was examined
for each purified enzyme (Table II). All four enzymes
were active toward N-fructosyl
N
-t-Boc-lysine as well as
fructosyl propylamine. However, the relative activity of amadoriases
Ia, Ib, and Ic toward fructosyl adamantanamine was low compared with
that for fructosyl propylamine. They had no activity against
N
-fructosyl
N
-acetyllysine. In contrast,
amadoriase II was active toward fructosyl adamantanamine and
N
-fructosyl
N
-acetyllysine. None of them were
active toward reduced fructosyl propylamine (10 mM),
glycated BSA (2 mg/ml), glycated poly-L-lysine (0.02%) or
glucose (10 mM). Apparent kinetic constants for
N
-fructosyl
N
-t-Boc-lysine and
fructosyl adamantanamine are shown in Table III. The
stoichiometry of the reaction catalyzed by the enzymes was determined
using fructosyl propylamine as a substrate. Stoichiometric consumption
of O2 and formation of H2O2 and
glucosone was observed for each enzyme (data not shown).
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In order to obtain information on the nature
of the cofactor, the absorption spectrum of amadoriase enzymes was
recorded. Fig. 7 shows the absorption spectra of
amadoriases Ib and II. Two major absorption bands were detected
at 362 and 452 nm with a shoulder at about 474 nm in the visible
region, indicating the presence of flavine as prosthetic group. The
spectra of the other enzymes were essentially the same.
pH Optimum and Isoelectric Points
The pH optima for amadoriases Ia, Ib, and Ic were pH 8.0 and for amadoriase II was pH 8.5. The isoelectric points of enzymes Ia, Ib, Ic, and II were 5.5, 5.5, 5.7, and 6.7, respectively.
Amino Acid SequencingTo identify the proteins, N-terminal sequencing was carried out using a gas-phase protein sequencer. The sequence of all four enzymes are shown in Table IV.
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We have isolated and purified four amadoriase enzyme fractions from Aspergillus sp. One of the four enzymes described above (amadoriase II) was different from the other three based on its N-terminal amino acid sequence and enzymatic properties. Amadoriase Ia and Ib appear identical, and amadoriase Ic have Michaelis-Menten properties and substrate specificities similar to amadoriase Ia and Ib. The N-terminal amino acid sequence of enzyme Ic is similar to that of enzymes Ia and Ib, except for the truncation of the first five N-terminal amino acids, which may have resulted from partial proteolytic breakdown during isolation. Thus, taken together, the data suggest the presence of two distinct amadoriase enzymes in the isolated Aspergillus strain. These enzymes cleave glycated substrates under consumption of oxygen and release of H2O2 and glucosone in a reaction involving FAD as a cofactor. Thus, they should be classified as fructosyl amine-oxygen oxidoreductases (EC 1.5.3). We propose the trivial name "amadoriase."
Search for sequence homology with other known proteins using the
combined GenBank CDS translations/PDB/Swiss Prot/SPupdate/PIR data base
with 220,000 sequences revealed that none of these proteins had been
described before. However, partial identity with D-amino acid oxidase from porcine (26) and human kidney (27), sti35 stress-inducible protein from Fusarium oxysporum (28), human p21 protein (29), and hypothetical 44.4-kDa protein in chromosomal I
from Schizosaccharomyces pombe is apparent, especially for
amadoriase Ic (Table IV). On close inspection, it turns out that all
the enzymes have the consensus sequence for the ADP-binding
-fold common to all FAD and NAD enzymes (30). All have the
hydrophobic residues that form the hydrophobic core between the
-strands and the
-helix.
The novel amadoriase enzymes described in this work join the growing
family of fructosyl amino acid oxidase enzymes that can deglycate low
molecular weight substrates under regeneration of the free amine while
producing H2O2 and glucosone. Table
V summarizes the currently known deglycating enzymes.
The first fully characterized enzyme was described by Horiuchi and
colleagues (31) in Corynebacterium sp., followed by a
similar enzyme from Aspergillus sp. by the same group (32).
Both enzymes regenerate the free amino acid and produce
H2O2 and glucosone. The first was obtained from
soil using fructosyl glycine as sole carbon and nitrogen source. It is
a dimeric protein with identical 44-kDa subunits, which degrades glycated amino acids with a Km value in the
submillimolar range. It has also activity against
D-erythropentulosylglycine and
D-tagatosyllysine but not against glycated alkylamines,
-amino acids, L-imino acids and D-amino
acids, N
-fructosyl lysine, or
-amino caproic acid. The Aspergillus enzyme was obtained
from the soil Aspergillus sp.1005 whose spores were grown in
a medium containing 1% fructosyl glycine. It is a dimeric protein of
83 kDa with identical 43-kDa subunits, i.e. similar to that
of the Corynebacterium enzyme but significantly different from our strain. The fungal enzyme had activity against many glycated amino acids, including N
-fructosyl
lysine, but, in contrast to the enzymes described in this work, had no
activity against glycated alkylamines. The Km for
fructosyl glycine was 2.2 mM, i.e. in the same
range as for our enzymes. The inability of Horiuchi's enzymes to
deglycate the Amadori products of alkylamines apparently relates
to the selection method.
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Recently, Sakai et al. (23) isolated a fructosyl lysine
oxidase from the fungus Fusarium oxysporum using
N-fructosyl
N
-Z-lysine as a sole
nitrogen source. The monomeric enzyme has, like our enzymes, a
molecular mass of 50 kDa. The Km for
N
-fructosyl lysine was very low (0.22 mM),
but little activity was observed against fructosyl valine. A small
amount of activity against glycated poly-L-lysine but not
against glycated protein was observed. In contrast to the two other
enzymes of Horiuchi, fructosyl lysine oxidase had covalently bound
FAD.
Finally, Genzyme scientists described in a European patent (33) the presence of deglycating enzymes in the bacterial groups Klebsiella and Corynebacterium, the fungi Acremonium and Fusarium, and the yeast genus Debaryomyces grown in media containing glycated butylamine as sole nitrogen source. All enzymes obtained generated glucosone and H2O2. One enzyme isolated from Debaryomyces vanrijiae had a Km of 80 µM for glycated butylamine, a pH optimum from 7.0 to 8.5, and highest stability between pH 5.0 and 7.5. Molecular weights of these enzymes are undetermined.
In spite of the failure to induce soil organisms to produce enzymes with activity against glycated proteins, the discovery of inducible amadoriase isoenzymes in our fungus, together with the reports above describing the presence of amadoriase enzymes in several genetically unrelated organisms such as prokaryotes, fungi, and yeast, raises the important question of the evolutionary significance of deglycating enzymes. Except for the Pseudomonas enzyme described earlier (17), whose isolation was recently described (34), all amadoriase enzymes that regenerate the intact amine also generate the highly reactive molecules glucosone and H2O2. In view of the growing number of data which directly implicate H2O2, oxidative stress, and Maillard reaction intermediates in diabetes (35, 36), the question thus arises as to whether amadoriase enzymes occur in human tissues. The sequence homology between amadoriase isoenzymes and mammalian D-amino acid oxidase suggests that deglycating or related enzymes could occur in the human. To our knowledge there has been no systematic investigation of that question, except for a previous short report, the conclusions of which were questioned (37, 38). Cloning of amadoriase cDNA, which is in progress in our laboratory, will provide important information for future research in the possible occurrence of fructosyl amino acid oxidase in mammalian tissues.
We thank Anne Morrissey, Laboratory of Clinical Microbiology, University Hospitals of Cleveland, for help with the identification of the soil organism.