©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Novel Degradation Pathway of Glycated Amino Acids into Free Fructosamine by a Pseudomonas sp. Soil Strain Extract (*)

(Received for publication, October 17, 1994)

Chiara Gerhardinger(§)(¶) M. Susan Marion(§)(**) Aleksandr Rovner Marcus Glomb Vincent M. Monnier(**)(§§)

From the Institute of Pathology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A Pseudomonas sp. soil strain, selected for its ability to grow on -(1-deoxyfructosyl) aminocaproic acid, was induced to express a membrane-bound enzymatic activity which oxidatively degrades Amadori products into free fructosamine. Apparent K values for fructosyl aminocaproate, -fructosyl lysine, fructosyl glycine, and ribated lysine were 0.21 mM, 2.73 mM, 3.52 mM, and 1.57 mM, respectively. The enzyme was also active against alpha-fructosyl lysine and borohydride-reduced Amadori product, weakly active with ribated and glycated polylysine, and inactive with reducing sugars, amino acids, and glycated proteins. The enzymatic activity was highest at pH 6.5 and 25 °C in 0.1 M sodium phosphate, while over 80% of the activity was lost above 65 °C. Complete inhibition was observed by HgCl(2), NaN(3), and NaCN suggesting a role for SH groups and copper in the enzymatic activity.

The reaction products were characterized by ^1H NMR, C NMR, and GC/MS and found to correspond to 1deoxy-1-aminofructose, i.e. free ``fructosamine,'' and adipic acid. Confirmation of the free fructosamine structure was based on the complete spectroscopic identity of the borohydride reduction product with commercially available glucamine (1-amino-1-deoxyglucitol). The new enzyme is provisorily classified as fructosyl N-alkyl amino acid oxidase (EC 1.5.3) (fructosyl-amino acid:oxygen oxidoreductase) and may thus belong to a novel class of ``Amadoriases'' which deglycate Amadori products oxidatively. In contrast, however, the new enzyme acts on the alkylamine bond rather than the ketoamine bond of the Amadori product.


INTRODUCTION

The recent results of the Diabetes Control and Complications Trial have brought new strength to the tenet that hyperglycemia plays a major role in the development of diabetic complications(1) . Currently, four mechanisms implicating glucose are under investigation. These include activation of the aldose reductase pathway(2) , altered gene expression(3) , altered signal transduction due to diacylglycerol formation(4) , and Maillard and oxidation reactions(5, 6) . More direct evidence in support of a role for hyperglycemia in the pathogenesis of diabetic complications by nonenzymatic glycation is the demonstration that levels of Maillard reaction products correlate with cumulative severity of diabetic complications (7) and the degree of glycemic control in dogs(8) . A role for advanced glycation in diabetic complications is also suggested by virtue of the fact that aminoguanidine, an inhibitor of glucose-mediated protein cross-linking both in vitro and in vivo(9, 10) , has beneficial effects on the progression of nephropathy(11) , retinopathy(12) , neuropathy(13) , and a host of diabetic processes (14, 15) in diabetic rodents. There is currently some controversy as to whether these effects result from scavenging of highly reactive Maillard reaction intermediates or from inhibition of nitric oxide synthase(16) . Yet, other experiments have shown that in vivo infusion of AGE proteins leads directly to increased capillary leakage in normal rat and that this process is also dependent on nitric oxide release(17) . Thus, these data suggest that pharmacological inhibition of the advanced Maillard reaction may have beneficial effects on the progression of hyperglycemia-related pathologies in diabetes.

The first major modification from which most subsequent advanced glycation compounds are formed is a ketoamine originating through Amadori rearrangement of the Schiff base initially formed by the reaction of glucose with protein amino groups. It would appear that inhibition of the glycation cascade at the level of the Amadori product should allow us to fully understand the role of nonenzymatic glycation in diabetes and aging. Although the formation of the Amadori product is slowly spontaneously reversible(18, 19) , it is presumably not fast enough to prevent the deleterious effects of glycation. In addition, since multiple mechanisms are thought to contribute to diabetes- and age-related pathologies, the precise role of the Maillard reaction in these complications is difficult to assess. For that reason we have searched for an enzyme with Amadori splitting activity from a soil organism capable of utilizing Amadori product for growth(20) . A Pseudomonas aeruginosa sp. was isolated from which a cytosolic protein which binds low molecular weight Amadori products in free form was purified to homogeneity and characterized(20) . That protein, however, had no Amadori product degrading activity. Searching for such activity, we now describe, in this strain, the presence of membrane-bound enzymatic activity which decomposes Amadori products into free fructosamine and adipic acid under utilization of oxygen.


EXPERIMENTAL PROCEDURES

Materials

D-Glucamine and N,O-bis(trimethylsilyl)acetamide (BtmSA) (^1)and thionyl chloride were obtained from Fluka. 2,3,5-Triphenyltetrazolium chloride (TTC), catalase from bovine liver, o-dianisidine dihydrochloride, and peroxidase from horseradish were obtained from Sigma. Adipic acid was from Aldrich. Silica gel-coated aluminum thin layer chromatography plates (0.2-mm thick) were obtained from EM Separations (Gibbstown, NJ). Whatman 17 chromatography paper was obtained from Whatman Inc. D-Glucosone was prepared by Dr. Marcus Glomb.

Microorganisms and Culture Media

A Pseudomonas sp. strain was isolated from soil by direct selection on minimal medium containing a synthetic Amadori product (fructosyl aminocaproate) as the only carbon source as described previously(20) . The medium was a minimal salt (KH(2)PO(4), 3 g/100 ml; K(2)HPO(4), 7 g/100 ml; (NH(4))(2)SO(4), 0.1 g/100 ml; 1 mM MgSO(4)) supplemented with biotin and thiamin, 0.01 mg/ml; riboflavin, 2.5 mg/ml; folic acid, pyridoxine, and pantothenic acid, 0.5 mg/ml; and fructosyl aminocaproate, 5 mg/ml. Batch cultures (>1 liter) were grown in nutrient broth containing fructosyl aminocaproate (1 mg/ml). Control media were either minimal media containing glucose and -aminocaproic acid or nutrient broth. Control bacterial species were obtained from the Clinical Microbiology Laboratory at the Institute of Pathology.

Synthesis of Glycated Substrates

Fructosyl aminocaproic acid and ribated lysine were synthesized as described previously(20) . -Fructosyl lysine and alpha-fructosyl lysine were, essentially, prepared according to Finot and Mauron(21) , and fructosyl glycine was a gift from Dr. Tatsuo Horiuchi (Kikkoman Corp., Noda, Japan). Glycated bovine serum albumin, glycated ribonuclease (RNase), and glycated poly-L-lysine were prepared as follows: bovine serum albumin, RNase, and poly-L-lysine (all from Sigma) were dissolved in 0.25 M sodium phosphate buffer (pH 7.4) at a final concentration of 50 mg/ml. Glucose or ribose was added to each protein solution to reach 1 M and 0.2 M, respectively. The solutions were filtered (0.2-µm filter; Millipore) and incubated at 37 °C for 30 days except the ribose-poly-L-lysine sample which was incubated for 2 days. Solutions were dialyzed twice against 0.05 M sodium phosphate buffer (pH 7.4) and stored at -20 °C until used.

Assay for Enzymatic Activity toward Amadori Products and Other Substrates

The enzymatic activity was assayed polarographically with a Clark type oxygen electrode (Beckman) at 25 °C. The assay mixture continuously stirred within the electrode chamber consisted of 100 µl of bacterial extract in 1.3 ml of 0.1 M sodium phosphate buffer (pH 7.4). After equilibration, the standard reaction was started by the addition of 20 µl of 0.7 M Amadori product solution. More dilute Amadori product concentrations were used for determination of kinetic parameters. Oxygen consumption was recorded and converted to micromoles of O(2) consumed/min as described by Robinson and Cooper(22) . All substrates were tested at 15 mM concentration (Table 1). Enzyme inhibitors were tested at a concentration of 7 mM (Table 2).





Preparation of Bacterial Extract

Crude extract from Pseudomonas sp. soil strain or from control strains, P. aeruginosa and Escherichia coli, was prepared as follows: cells from 24-h cultures (500 ml to 4 liters) were harvested by centrifugation at 5000 times g, washed once with minimal salt, resuspended in sodium phosphate buffer ((pH 7.4) 10 ml/liter culture)), 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, and disrupted by three passes through a French press at 6000-7000 p.s.i. followed by sonication. The cell lysate was then centrifuged at 3000 times g to remove unbroken cells, and the supernatant was tested for Amadori product degrading activity under the standard assay conditions. Protein concentration was determined by the method of Bradford(23) .

Preparation of Membrane-rich Fraction

All steps were performed at 4 °C. Cells harvested from 4 liters of nutrient broth, supplemented with fructosyl aminocaproate, were washed and disrupted as above. The extract (500-600 mg of total proteins) was brought to 30% ammonium sulfate saturation and centrifuged at 15,000 times g for 15 min. The precipitate was resuspended with 10 ml of 0.05 M sodium phosphate buffer, pH 7.4, containing 0.2 mM phenylmethylsulfonyl fluoride, and dialyzed twice against the same buffer. The sample was centrifuged at 33,000 times g for 3 h, and the pellet was resuspended in a minimal amount of buffer and stored at 4 °C.

Evaluation of the Optimum Reaction Conditions

Enzymatic activity was characterized in relation to pH, temperature, and stability. The assays were performed with fructosyl aminocaproate as substrate under standard conditions. The pH optimum was determined with various buffers (sodium phosphate, sodium citrate, Tris-HCl, glycine-NaOH, all 0.1 M) at pH ranging from 3.5 to 10.6. The effect of temperature was evaluated by keeping the reaction mixture at various temperatures for 15 min. The solutions were immediately chilled on ice, and the activity was measured.

Identification and Purification of Enzymatic Degradation Products

For identification of the product, the reaction mixture contained 1.5 µmol of sodium phosphate buffer (pH 8.0), approximately 8 mg of washed ammonium sulfate-precipitated soil organism extract, and 13.7 µmol of fructosyl aminocaproate in a total volume of 170 µl. The reaction was performed at 25 °C or 37 °C for 5 days. Control experiments were either extract or Amadori product only. At designated time points, samples were centrifuged at 18,000 times g for 15 min and spotted onto TLC plates, along with -aminocaproate, glucosone, and glucose. The plates were developed with a solvent system of n-butanol/acetic acid/water (5:3:1). Dried plates were sprayed with either ninhydrin (0.2%) or TTC (2% in 0.5 M NaOH). TTC is specific for reducing compounds, and reducing sugars react with the reagent to give a bright red spot. For purification of the TTC positive material, supernatants from a number of reaction mixtures (25 °C) were applied to Whatman 17 chromatography paper, developed with n-butanol/acetic acid/water (9:2:5), and the product was eluted from the paper with water and lyophilized. Detection of adipic acid by TLC was done utilizing the same TLC plates as for fructosamine and a solvent system consisting of di-isopropyl ether, formic acid, and water (90:7:3). Upon spraying with bromcresol green reagent and heating, standard adipic acid gave a bright yellow spot (R(f) = 0.6) on a green background. Co-chromatography between standard and experimental samples was observed only after desalting of the sample over Dowex 50 in HCl form.

Spectroscopy

All test substances for NMR spectroscopy were exchanged once with D(2)O, dissolved in D(2)O, and scanned with a 300-MHz NMR spectrometer (Gemini-300, Varian Associates, Inc. Palo Alto, CA). The TTC-positive enzymatic reaction product and the Amadori product were reduced with 3 and 10 times more molar excess NaBH(4) in 0.01 N NaOH for 1 h, respectively. TLC confirmed complete reduction. Following neutralization with HCl and lyophilization, samples were passed through three changes of 1% methanolic HCl and centrifuged to remove borohydride and salt. Standard glucamine was also neutralized with HCl and treated with 1% MeOH-HCl. All samples for ^1H NMR and C NMR were submitted to approximately 100 scans and 3600 scans (glucamine to 100 scans), respectively.

The dried reduced reaction product and glucamine were converted to trimethylsilyl derivatives by treating with BtmSA/pyridine (4:3) for 60 min at 25 °C(24) . 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, inside diameter 0.2 mm, film thickness 0.33 µm; Hewlett-Packard Co.). Methane was used for positive chemical ionization. Injector and interface temperatures were 270 °C and 280 °C, respectively. Temperature program was 100 °C to 200 °C at 5 °C/min, 200 °C to 270 °C at 10 °C/min, isothermal at 270 °C for 10 min. For confirmation and quantitation of adipic acid as the enzymatic split product of fructosyl aminocaproate, the material obtained after 5 days of incubation was subjected to TLC, as described before, and GC/MS with Electron Impact ionization (70 eV). Standard adipic acid (1 mg) and desalted dry experimental samples from 180 µl of enzyme supernatant were dissolved in 1.0 ml of methanol and evaporated three times to remove excess water. To 0.5 ml of solution were added 10 drops of thionyl chloride, and heating was carried out for 60 min at 100 °C. Unreacted thionyl chloride was evaporated three times with 0.5 ml of MeOH. The dry residue was taken up in 200 µl of ethyl acetate and passed over a 1.5-cm silica gel column in a Pasteur pipette and eluted with 1.8 ml of ethyl acetate. This fraction was concentrated to 25 µl, and 1 µl was injected. Temperature program was 100 °C to 200 °C at 5 °C/min, 200 °C to 270 °C at 10 °C/min, and isothermal at 270 °C for 10 min.

Quantitative Time Course Experiment

Formation of free fructosamine and adipic acid from fructosyl aminocaproate was followed over time. Adipic acid was quantitated by GC/MS as described above, and fructosamine was quantitated by HPLC as glucamine following sodium borohydride reduction. HPLC analysis was performed on a Waters gradient system (Waters Chromatography Division) consisting of two model 510 pumps and a model 470 fluorescence detector with o-phthalaldehyde post-column detection (31) and a C-18 reverse phase column (VYDAC 218TP54, The Separations Group). Solvent A was 5% n-propyl alcohol, and solvent B was 60% n-propyl alcohol, both containing 3 g of SDS, 1 g of NaH(2)PO(4)bulletH(2)O in 1 liter with pH adjusted to 2.8 with H(3)PO(4). Flow was 1.0 ml/min, and the program was 15% B for 0-10 min, 100% B at 15 min until 25 min, and 15% B for 20 min prior to injection. Under these conditions, standard glucamine eluted at 9.6. min, and -aminocaproic acid eluted at 16.0 min.

For the time course experiment, 100 µl of Pseudomonas extract was incubated for 0, 24, 48, 72, and 120 h with 80 µl of phosphate-buffered saline and 140 µl of Amadori solution to make the final concentration 100 mM. The work-up for the adipic acid determination is described above. For glucamine determination, 250 µl of solution were reduced with 50 mg of NaBH(4) in 1 ml of 0.01 N NaOH for 60 min, acidified with 150 µl of 6 N HCl, treated three times with methanolic HCl to remove borate salts, and taken up in 2 ml of H(2)O from which 50 µl were injected into the HPLC system.


RESULTS

Identification of Amadori Product Degrading Activity in Extract from Pseudomonas sp. Soil Strain

A preliminary experiment revealed that incubation of Pseudomonas sp. soil strain extract with lysine-Sepharose glycated with [^14C]glucose was followed by release of radioactivity into the supernatant (not shown), suggesting the possible presence of enzymatic activity. Further experiments revealed that oxygen was consumed when extract from the bacterium, grown in media containing fructosyl aminocaproate, was incubated with this Amadori product. To confirm the specificity for Amadori product, the oxygen consumption assay was performed using extract from the soil organism grown in minimal medium containing both glucose and -aminocaproic acid or nutrient broth only. In both cases, only minor activity was detectable suggesting that a substrate-inducible enzyme was degrading Amadori products. Extract fractionation by ammonium sulfate precipitation and high speed centrifugation showed that enzymatic activity was retained in the precipitate. The likelihood that the Amadori product degrading activity was membrane-bound was suggested by an experiment in which the insoluble pellet from the French press was further fractionated by differential centrifugation in the presence of 0.5% deoxycholate. Substantial activity was recovered in the middle layer of a 15/70% sucrose step gradient obtained at 140,000 times g for 60 min.

Substrate Specificity and Kinetic Constant

To investigate the substrate specificity, several compounds were tested. As shown in Table 1, significant oxygen consumption was observed with the free Amadori products, while no activity was observed with the unglycated amines and sugars. Surprisingly, 20% activity toward borohydride-reduced Amadori product of -aminocaproate was observed. In order to rule out an effect due to incomplete reduction, glucitolyl -aminocaproate was synthesized according to the method of Schwartz and Gray (30) using sodium cyanoborohydride. Again, significant activity was noticed. Of the glycated proteins tested, only ribated-poly-L-lysine was found to have activity. When the assay was performed with increasing substrate concentrations, a typical MichaelisMenten curve was obtained as shown in Fig. 1for fructosyl aminocaproate and fructosyl lysine, respectively. Apparent K(m) and V(max) were 0.21 mM and 0.028 µmol of O(2)/min for fructosyl aminocaproate, 2.73 mM and 0.021 µmol of O(2)/min for fructosyl lysine, 3.52 mM and 0.017 µmol of O(2)/min for fructosyl glycine, and 1.568 mM and 0.013 µmol of O(2)/min for ribated lysine.


Figure 1: Michaelis-Menten and Lineweaver-Burk plots for fructosyl aminocaproate (A) and -fructosyl lysine (B). The enzymatic activity was measured at room temperature in 0.1 M sodium phosphate buffer (pH 7.4).



Optimum Reaction Conditions

As shown in Fig. 2A, maximum activity was observed at pH 6.5 in sodium phosphate buffer and pH 5.5 in citrate buffer. Activity was greatly reduced at pH values below 4.5 and above 9.0. In the range of temperature tested, the highest activity was at 25 °C, decreasing to 35% at 45 °C, and less than 20% at 65 °C (Fig. 2B). Heating at 100 °C for 10 min resulted in complete inactivation of the enzyme.


Figure 2: Effects of pH and buffer composition (A) and temperature (B) on the enzymatic activity. circle, 0.1 M citrate; bullet, 0.1 M sodium phosphate; up triangle, 0.1 M Tris-HCl; , 0.1 M glycine-NaOH. The activity is expressed as percent of the control (i.e. the activity in 0.1 M sodium phosphate buffer at pH 7.4 and 25 °C).



Effect of Chemicals and Metal Ions

All compounds tested were added to the assay mixture prior to the addition of fructosyl aminocaproate. Metal ions such as Mn, Mg, Zn, and Ca did not interfere with the reaction at 7 mM concentration (Table 2). Moreover, neither a chelating agent, EDTA, nor a disulfide bond reducing agent, dithiothreitol, was found to inhibit or increase oxygen consumption. However, enzymatic activity was completely lost in the presence of NaN(3), HgCl(2), and NaCN and decreased to 35% and 50% with CuSO(4) and AgNO(3), respectively. Since Hg, Cu, and Ag ions are good ligands of thiol groups, this suggests that free thiols are required for enzymatic activity. In addition, TLC showed increased degradation of Amadori product in the presence of dithiothreitol. Since azide and CN, known inhibitors of cytochrome oxidases (25) and a number of copper-containing oxidases (26) , inhibited the activity, it is likely that the Amadori product oxidase requires copper for activity. The latter, however, is firmly bound to the protein since the activity was not lost in the presence of EDTA.

The enzymatic activity was labile to detergents of all classes, i.e. ionic, nonionic, and zwitterionic. Triton, sodium dodecyl sulfate, and glucoside completely inactivated the enzyme, while, with deoxycholate, methylglucamides, and zwitterionic detergents, some activity was detectable (data not shown).

Identification, Isolation, and Characterization of Reaction Products

The disappearance of fructosyl aminocaproate and formation of a new product was monitored by TLC. Following 2 h of incubation, a product which reacted with both ninhydrin (producing yellow color) and TTC could be detected at an R(f) of 0.24 (Fig. 3). By 5 days, fructosyl aminocaproate was completely degraded. The new product was not detected in reaction mixtures which contained only fructosyl aminocaproate or only Pseudomonas membrane extract. The new product was not -aminocaproate, glucosone, or glucose since these did not react with both ninhydrin and TTC, nor did they have comparable R(f) values. In contrast, the borohydride-reduced reaction product was chromatographically similar to glucamine in that both reacted only with ninhydrin (producing a blue color) and both migrated to an R(f) value of 0.18 (not shown).


Figure 3: TLC of time course experiments. Reaction of Pseudomonas sp. soil strain extract with -(1-deoxyfructosyl) aminocaproic acid (Extract + DF - ACA) resulted in the formation of a new ninhydrin (NIN) and TTC-positive product (R 0.24) and the complete degradation of the Amadori product by 5 days. The major reaction product was different from -aminocaproic acid (ACA), glucosone (GLN), or glucose (GLU) and co-migrated with glucamine upon borohydride reduction (not shown).



The TLC data showing the presence of a new ninhydrin-positive product with reducing properties together with the ^1H NMR data strongly suggested that the Amadori product had been cleaved on the N-alkyl side of the Amadori product instead of the ketoamine bond as described previously(28, 29) . The putative breakdown product was expected to be 1-amino-1-deoxyfructose, i.e. free fructosamine. In order to confirm this presumption, the product was prepared quantitatively using paper chromatography and characterized spectroscopically. Comparison of the ^1H NMR spectrum of the substrate fructosyl aminocaproate (Fig. 4A) with the spectrum of the product (Fig. 4B) showed loss of proton signals < 3 ppm that represent the methylene protons of caproic acid in the Amadori product. However, there was retention of the singlet at about = 3.1 ppm which is attributed to the noncoupling methylene protons on C-1 of the sugar. The product was reduced with sodium borohydride and compared with commercially available glucamine (1-amino-1-deoxyglucitol) in terms of ^1H NMR, C NMR, and GC/MS properties. The ^1H NMR spectra of the reduced reaction product and commercial compounds were identical (Fig. 5, A and B) and revealed the disappearance of signals due to methylenic protons of caproic acid and the singlet at = 3.1 ppm. Minor signals representing contaminants were present at = 3.2-3.4 and = 3.7-3.8 ppm.


Figure 4: ^1H NMR spectrum of the purified product. TTC and ninhydrin-positive degradation product of fructosyl aminocaproate. Comparison of the spectrum of fructosyl aminocaproate (A) with the spectrum of the reaction product (B), both in D(2)O, showed the absence of caproic acid protons ( < 3 ppm) in the reaction product while the remaining signals of degradation (B) are virtually identical with the ``sugar'' signals of native Amadori product.




Figure 5: ^1H NMR spectra of NaBH(4)-reduced product and glucamine. The spectrum of glucamine (A) is identical with that of the reduced reaction product (B). Signals due to minor contaminants are present at = 3.2 - 3.4 ppm and = +3.7 - 3.8 ppm.



Comparison of the attached proton test C NMR spectra of glucamine (Fig. 6A) and the reduced reaction product (Fig. 6B) again suggested that the borohydride reduction product was glucamine. The two major signals above baseline are due to C-1 and C-6, as they represent secondary carbons with two methylene protons each, in contrast to C-2 through C-5 which are tertiary carbons with only one proton attached. The latter result in the four major signals below baseline. The minor peaks in the spectrum of the reaction product are due to impurities not removed during isolation.


Figure 6: Attached proton test C NMR spectra of glucamine (A) and borohydride-reduced ninhydrin and TTC positive product (B). The spectrum of glucamine was nearly identical with that of the reduced reaction product (B), both in D(2)O. Asterisks (*) indicate the presence of impurities not removed during product isolation.



Confirmation of the identity of the reaction product was achieved by GC/MS. Trimethylsilyl derivatives of the reduced product and glucamine were prepared with BtmSA. The retention times of 22.97 min were identical for the synthetic and experimental product (Fig. 7A, shown here for standard only). In addition, two minor signals are also found in the chromatogram of the experimental sample at 27 and 32.8 min. The fragmentation patterns of the chemical ionization mass spectra of the two trimethylsilyl derivatives were identical for all major and minor signals (Fig. 7, B and C, lower profile). The M + 1 peak at m/z 614 concurs with the actual molecular weight of 181 for glucamine plus 6-fold derivatization by trimethylsilyl groups. The two major signals at m/z 542 and 526 correspond to losses of (CH(3))(2) Si=CH(2) and NH(2)-trimethylsilyl, respectively.


Figure 7: GC/MS spectra of trimethylsilyl derivatives of standard glucamine (A) and borohydride-reduced enzymatic breakdown product (B). The reduced reaction product as well as glucamine standard eluted in a double peak at 22.97 min. The double peaks are attributed to epimers. The fragmentation pattern of the chemical ionization spectrum of the reduced enzymatic degradation product is identical with that of glucamine.



All data so far concurred with the presence of a split of the Amadori product at the N-alkyl bond of -aminocaproic acid. Therefore, one would expect to detect the caproic aldehyde (6-oxohexanoic acid) which, however, would quickly oxidize to adipic acid. Adipic acid was indeed detected by both TLC (not shown) and GC/MS using the methyl ester derivative and thionylchloride method (Fig. 8, A and B). The fragmentation spectrum obtained by electronic ionization shows, among other signals, typical alpha-fragmentation of an ester with m/z 59 and 143, as well as M/2 signal at m/z 87 which is common for a symmetric molecule.


Figure 8: GC/MS spectra of methyl esters of standard adipic acid (A) and adipic acid recovered from the enzymatic degradation of glycated -aminocaproate (B). Retention time was 7.63 and 7.64 min, respectively. The electron ionization spectra are identical and show the typical m/z 143 signal corresponding to the loss of one methoxy group.



A time course experiment on the formation of free fructosamine and adipic acid was carried out (Fig. 9). Both concentrations increase as a function of time, whereby a moderately higher recovery was obtained for fructosamine than adipic acid. The difference may be due to a partial loss of adipic acid during sample preparation which was performed without added internal standard. Throughout the incubation period, there was no change in the peak height of -aminocaproate.


Figure 9: Time course formation of free fructosamine () and adipic acid (up triangle) in incubations of fructosyl aminocaproate (100 mM) and an insoluble extract of P. aeruginosa soil organism. Fructosamine was assessed as glucamine by HPLC and adipic acid in the form of methyl ester by GC/MS.



Characterization of Reduced Electron Acceptor

Hydrogen peroxide was not detected in the reaction mixtures by either of two methods, the oxygen electrode method or the chromogenic method. In the first method, catalase (24,000 units) was added, and oxygen production was measured with the oxygen electrode. In the second method, peroxidase and o-dianisidine were added, and oxygen production was measured indirectly as oxidized o-dianisidine, a colored compound(27) .


DISCUSSION

This work was initiated with the goal of finding an enzyme that would be potentially useful for in vivo reversal of nonenzymatic glycation as a tool for testing specifically the role of the Maillard reaction in the complications of diabetes and aging. For that reason, we began to search for Amadori product degrading enzymes in soil organisms utilizing an Amadori product as the sole carbon source for growth(20) . While this work was in progress, two enzymes with Amadori product degrading activity were isolated and characterized by Horiuchi and co-workers (28, 29) using a similar approach.

The enzyme described in this study has some features in common with that of Horiuchi and co-workers(28, 29) , in that it appears to be membrane-bound, to degrade Amadori products oxidatively, and to have poor or no activity toward glycated proteins. The major difference lies in the cleavage site of the Amadori substrate which occurs not at the ketoamine bond, but at the alkylamine bond of the glycated amino acid (Fig. 10). In addition, our enzyme did not generate H(2)O(2). Based on the negative reaction for aldehydes (TTC) on the TLC plate (Fig. 3), we suspected and confirmed that the expected initial product 6-oxohexanoic acid was rapidly oxidized to adipic acid. In contrast, the enzyme described by Horiuchi and co-workers (28, 29) released glucosone.


Figure 10: Reaction scheme. Comparison of the novel degradation pathway of glycated -aminocaproic acid by Pseudomonas extract (A) and fructosyl amino acid oxidase from Corynebacterium (B).



The fact that our enzyme cleaved the amino alkyl bond instead of the ketoamine bond is unexpected since isomerization of the ketoamine bond into the imine, which is necessary for nucleophilic attack, would appear to be much more favored than isomerization of the amino alkyl bond. The mechanism of action, however, may be more complex, since the enzyme also had activity against borohydride-reduced Amadori product.

Since the experiments were carried out with a membrane fraction instead of a pure enzyme, it could be argued that the novel degradation pathway of Amadori product is, in fact, the product of two enzymes, e.g. a isomerase reverting the Amadori product to glucose/mannose, followed by a deaminase which would generate 6-oxohexanoic acid and ammonia. The latter would then react with the glucose/mannose to form free fructosamine. To rule out this possibility, control experiments were carried with membrane extract incubated with either free -aminocaproate, ammonia and glucose, or glucosone. However, none of these experimental conditions led to the formation of free fructosamine. We therefore conclude that the degradation of Amadori product into free fructosamine and adipic acid is the result of a novel enzyme that requires the intact substrate to act on.

Preliminary investigation of the nature of the cofactor showed that the Pseudomonas enzymatic activity was inhibited by Hg, Cu, and NaN(3), similarly to the enzyme isolated from the Aspergillus sp. strain by Horiuchi and Kurokawa (29) while the oxidase isolated from the Corynebacterium sp. (28) was insensitive to the latter compound. The previously isolated enzymes were both shown to be FAD-dependent enzymes. The observation that the Pseudomonas enzyme was inhibited by CN strongly suggests that copper is involved in the active site. Studies with the pure enzyme will be necessary to identify the coenzyme required for the transfer of the electron to oxygen.

Preliminary efforts to purify the enzyme using the solubilization procedure of Horiuchi and co-workers (28, 29) and commercially available detergent kits showed that more than 80% of the activity is lost once the enzyme is released from the membrane-rich fraction. Thus, its molecular weight is yet unknown. In the case of the fructosyl amino acid oxidase from Corynebacterium sp. and the fructosyl amine oxidase from Aspergillus sp., the enzymes had a M(r) = 44,000 and 43,000 on SDS-polyacrylamide gel electrophoresis, respectively. The enzyme from Corynebacterium sp. had a M(r) = 88,000 when chromatographed on Sephadex G-200 suggesting dimerization. Although the Amadori product binding protein which was isolated from the same microorganism by affinity chromatography had a similar molecular weight (M(r) = 45,000), this protein showed no enzymatic activity.

From an application viewpoint, the ``Amadoriases'' described by us and Horiuchi and co-workers (28, 29) could be useful diagnostic tools for determining the extent of glycation in samples containing amino acids or proteins. In the latter case, the protein substrates would need to be pretreated with proteases and then reacted with the enzyme. It is conceivable that Amadoriases which act directly on proteins could be obtained through appropriate selection of soil organisms or through genetic engineering of existing enzymes.


FOOTNOTES

*
This project was supported by grants from the Juvenile Diabetes Foundation International and in part by National Institute on Aging Grant AG 05601 and National Eye Institute Grant EY 07099. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
The first two co-authors contributed equally to the work described in this article.

Recipient of a fellowship from the Juvenile Diabetes Foundation International. Current address: Schepens Eye Inst., Harvard Medical School, Boston, MA 02114.

**
Supported by National Inst. on Aging Grant T32AG00105.

§§
To whom correspondence and reprint requests should be addressed. Tel: 216-368-6613; Fax: 216-844-1810.

(^1)
The abbreviations used are: BtmSA, N,O-bis(trimethylsilyl)acetamide; fructosyl aminocaproate, -(1-deoxyfructosyl) aminocaproic acid; fructosyl glycine, 1-deoxyfructosyl glycine; fructosyl lysine, alpha-t-Boc--(1-deoxyfructosyl) lysine; ribated lysine, alpha-t-Boc--(1-deoxyribosyl) lysine; HPLC, high performance liquid chromatography; TTC, triphenyltetrazolium chloride.


ACKNOWLEDGEMENTS

We thank Dr. Robert Hogg, Dept. of Microbiology and Molecular Biology, for helpful discussions throughout this work, and Dr. Amit K. Saxena for performing the inhibition experiment with CN. We also thank Dr. Michael Jacobs and the laboratory for Clinical Microbiology at the Institute of Pathology for their assistance throughout this project.


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