(Received for publication, October 17, 1994)
From the
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
-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
, NaN
, and NaCN suggesting a role for SH
groups and copper in the enzymatic activity.
The reaction products
were characterized by H 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.
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.
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.
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 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
O from which 50 µl were injected into the HPLC system.
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).
Figure 2:
Effects of pH and buffer composition (A) and temperature (B) on the enzymatic activity.
, 0.1 M citrate;
, 0.1 M sodium phosphate;
, 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).
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).
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 H 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
H 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
H
NMR,
C NMR, and GC/MS properties. The
H 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:
H 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
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:
H NMR spectra of
NaBH
-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
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)
Si=CH
and NH
-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
-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
(
) 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.
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
HO
. 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
,
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 = 44,000 and 43,000 on SDS-polyacrylamide
gel electrophoresis, respectively. The enzyme from Corynebacterium sp. had a M
= 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
= 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.