Redox Regulation of Transglutaminase 2 Activity*

Transglutaminase 2 (TG2) in the extracellular matrix is largely inactive but is transiently activated upon certain types of inflammation and cell injury. The enzymatic activity of extracellular TG2 thus appears to be tightly regulated. As TG2 is known to be sensitive to changes in the redox environment, inactivation through oxidation presents a plausible mechanism. Using mass spectrometry, we have identified a redox-sensitive cysteine triad consisting of Cys230, Cys370, and Cys371 that is involved in oxidative inactivation of TG2. Within this triad, Cys370 was found to participate in disulfide bonds with both Cys230 and its neighbor, Cys371. Notably, Ca2+ was found to protect against formation of these disulfide bonds. To investigate the role of each cysteine residue, we created alanine mutants and found that Cys230 appears to promote oxidation and inactivation of TG2 by facilitating formation of Cys370–Cys371 through formation of the Cys230–Cys370 disulfide bond. Although vicinal disulfide pairs are found in several transglutaminase isoforms, Cys230 is unique for TG2, suggesting that this residue acts as an isoform-specific redox sensor. Our findings suggest that oxidation is likely to influence the amount of active TG2 present in the extracellular environment.

Human transglutaminase 2 (TG2) 2 modifies protein-or peptide-bound glutamine residues by either cross-linking their reactive carboxamide side chains to primary amines or by deamidation, converting glutamine residues to glutamate (1,2). Ca 2ϩ is required for this catalytic activity and induces conformational changes in the enzyme, which arrange the active-site residues for catalysis, including Cys 277 (3)(4)(5)(6). As TG2 is abundantly expressed in both the intracellular and extracellular environments of many tissues, its catalytic activity must be tightly regulated to avoid excess modification of cellular and tissue components. GTP and GDP act as allosteric inhibitors by inducing a closed conformation in which the active site is buried (6 -8). Low Ca 2ϩ concentration and high GTP/GDP concentration in the cytosol typi-cally prevent TG2 activation within cells. Despite conditions in the extracellular milieu that favor activation, extracellular TG2 also appears to be predominantly inactive under normal conditions but can be activated by certain types of inflammation and cell injury (9).
The catalytic activity of TG2 is implicated in the pathogenesis of several human diseases, including celiac disease (10). Celiac disease is caused by an aberrant immune response to proline-and glutamine-rich peptides from dietary gluten in the small intestine of genetically predisposed individuals (11). In the celiac immune response, the enzymatic activity of TG2 is crucial, as TG2-mediated deamidation of gluten peptides increases their T-cell antigenicity (12,13).
In contrast to our knowledge of the mechanistic basis for TG2 inactivity in the intracellular environment, the mechanisms underlying regulation of TG2 activity in the extracellular compartment remain unclear. TG2 harbors no disulfide bonds in its native state, which is unusual for enzymes in the extracellular environment (14). Previous studies have shown that TG2 is susceptible to oxidation, resulting in inactivation (19 -22). Thus, modulation of enzymatic activity through oxidation presents a plausible mechanism for regulation of extracellular TG2 activity. We have investigated the events underlying oxidative inactivation of TG2 and report the identification of a redox-sensitive cysteine triad consisting of Cys 230 , Cys 370 , and Cys 371 . Within this triad, Cys 230 appears to set the threshold for intramolecular disulfide bond formation and thereby inactivation. Oxidation was influenced by the presence of Ca 2ϩ and substrate, suggesting that the local environment can modulate and fine-tune oxidative inactivation of TG2 in the extracellular milieu.

EXPERIMENTAL PROCEDURES
TG2 Expression and Purification-Human wild-type (WT) TG2 3  in Escherichia coli BL21(DE3) cells as N-terminally His 6 -tagged proteins (pET-28a, Novagen, Madison, WI). Expression of TG2 was induced by isopropyl ␤-D-thiogalactopyranoside or OnEx Solution 1-3 (Novagen), followed by overnight incubation at 24°C for the WT enzyme and 12-18°C for mutants. The recombinant protein was purified by nickel-nitrilotriacetic acid affinity chromatography and anion exchange chromatography before dialysis and storage at Ϫ70°C. The human active-site TG2 mutant C277S, expressed in a baculovirus system and purified to homogeneity by Ni 2ϩ affinity chromatography, was a gift from Eckart Mummert (Phadia GmbH, Freiburg, Germany). Inhibitor Synthesis and Preparative Inhibition of TG2-The active-site inhibitor Ac-P(6-diazo-5-oxo-L-norleucine)LPF-NH 2 was synthesized and used to inactivate human TG2 as described previously (15).
TG2 Deamidation Kinetics-Kinetic parameters for deamidation of glutamine side chains in peptide substrates were measured using modifications of a method described previously (16). The components were as follows: 1) a concentrated buffer consisting of 1 M MOPS, 5 mM EDTA, and 50 mM ␣-ketoglutarate (pH 7.2); 2) 100 mM CaCl 2 ; 3) 300 mM benzyloxycarbonyl-Gln-Gly (Sigma) prepared as a solution of the sodium salt by titration with sodium hydroxide and diluted to 10ϫ stocks of the desired reaction concentrations; 4) 50 mM NADH; and 5) lyophilized ammonium-free glutamate dehydrogenase (Biozyme Laboratories) dissolved to 0.5 units/l in 200 mM MOPS, 1 mM EDTA, and 10 mM ␣-ketoglutarate (pH 7.2) and clarified by centrifugation. Each reaction contained 20 l of component 1, varying amounts of component 2, 10 l of component 3, 2.5 l of component 4, and an appropriate amount of water to reach 91.8 l. To this mixture was added 7.2 l of component 5. The resulting mixture was pre-equilibrated for 30 min at room temperature, and 97 l was added to a microtiter plate well. To initiate the deamidation reaction, typically 3 l of WT or mutant TG2 stock solution was added to each well. Absorbance was monitored at 340 nm and 30°C using a Molecular Devices SpectraMax Plus 384 microplate reader. Slopes were calculated from the linear regions of each trace, typically from 1600 to 3600 s. Ca 2ϩ concentrations reported are those of CaCl 2 added minus the EDTA concentration in the final reaction buffer. For a given experiment, the data for wildtype TG2 and all mutants were collected simultaneously to facilitate direct comparison. Rate constants were calculated using Origin 6.0.
Sample Oxidation and In-gel Digestion for Mass Spectrometric Analysis-Controlled oxidation was performed by treatment of WT or mutant TG2 with varying ratios of GSH and GSSG in N 2 -flushed 100 mM Tris-HCl (pH 7.2) and 0.5 mM EDTA. Typically, 0.1 or 0.05 g/l enzyme was incubated with 2 mM GSSG and 0.24, 0.45, 1.25, or 2.4 mM GSH in the presence or absence of synthetic peptide substrate (DQ2-␣-I, QLQPF-PQPQLPY; or DQ2-␣-II, PQPQLPYPQPQLPY-NH 2 ) and 0, 1, 2, 5, or 10 mM CaCl 2 . Oxidation was performed at 30 or 37°C and stopped at the indicated time points by precipitation of TG2 using trichloroacetic acid to a final concentration of 10% (w/v), followed by a 10-min centrifugation at 4°C and precipi-tation two times in 100% ice-cold acetone. Precipitated protein was resuspended and alkylated with 25 mM iodoacetamide (IAM; Sigma) in 1% SDS and 50 mM Tris-HCl (pH 8.0) at room temperature with shaking in the dark for 1 h, followed by nonreducing SDS-PAGE (12% Tris-HCl Ready Gel, Bio-Rad) and Coomassie Blue staining. Coomassie Blue-stained bands were cut out, destained, and washed with H 2 O, 1:1 H 2 O/acetonitrile, and 100% acetonitrile (three times for 20 min each). Gel pieces were either directly digested with trypsin (see below) or reduced with 10 mM DL-dithiothreitol (DTT; Sigma) and alkylated with 55 mM iodoacetic acid (IAA; Sigma), followed by washing and overnight trypsin digestion at 37°C.
Nondenaturing PAGE-After preincubation of proteins as indicated in the figure legends, samples were diluted in native Laemmli sample buffer without SDS and reducing agents and separated on 4 -20% Tris-HCl Ready Gel using Tris/glycine running buffer adjusted to pH 8.5 at 4°C. Gels were run at 125 V for 1 h at 4°C.
Identification of Disulfide Bridges and Quantification of Cysteine Oxidation by Mass Spectrometry-Trypsin-digested samples were analyzed by mass spectrometry (MALDI-ToFToF or Nano-LC-QToF system, Bruker Daltonics). Peptides harboring cysteine residues were identified by protein data base searches using the search engine Mascot (17) and by manual sequencing. Oxidation of cysteine residues was quantified by calculating the average mass shift of the isotopic envelope of peptides from control and oxidized samples alkylated with IAM and IAA. The relative oxidation of Cys 370 and Cys 371 was quantified as follows. The signal at m/z 1481.8 corresponds to the tryptic peptide in which both Cys 370 and Cys 371 were present in a reduced state and therefore were labeled with IAM (ϩ57 Da, ϩ57 Da). The signal at m/z 1482.8 derives from the tryptic peptide with one of the cysteine residues participating in disulfide bond formation. That cysteine residue was thus labeled with IAA instead of IAM (ϩ57 Da, ϩ58 Da). In addition, this signal harbored the 13 C isotopic contribution from the peak at m/z 1481.8 (78% of the intensity at m/z 1481.8). The signal at m/z 1483.8 consisted of oxidized Cys 370 and Cys 371 both labeled with IAA (ϩ58 Da, ϩ58 Da) plus the contribution coming from the isotopic envelope of the signals at m/z 1481.8 (43%) and m/z 1482.8 (78%). The isotope contribution from neighboring peaks was subtracted, and relative intensities were calculated as the intensity of one peak divided by the sum of all three peaks. This corresponds to the percentage of reduced and oxidized Cys 370 and Cys 371 present.
Determination of Enzymatic Activity of Oxidized Samples-Synthetic peptide substrates and Ca 2ϩ were added to give final concentrations of 20 -100 M peptide, 5 mM Ca 2ϩ , and 0.1 or 0.05 g/l enzyme and then incubated at 37°C. Aliquots were removed at the indicated time points. Deamidation of DQ2-␣-I or DQ2-␣-II was quantified by MALDI-TOF mass spectrometry.
Determination of Enzymatic Activity of Fibronectin-bound TG2-Microtiter 96-well plates were coated overnight with human fibronectin (25 g/ml in 50 mM bicarbonate buffer (pH 9.6); Roche Applied Science). Thereafter, TG2 (5 and 2.5 g/ml in Tris-buffered saline with 0.1% Tween 20 and 1 mM DTT) was immobilized for 1 h at 37°C, followed by incubation with 1 mM GSSG or 1 mM DTT for 1 h at 37°C. Enzymatic activity was measured by addition of 40 M 5-BP and 10 mM CaCl 2 . After extensive washing, incorporated 5-BP was detected with streptavidin-alkaline phosphatase. Absorption was measured at 405 nm after the addition of 1.5 mg/ml alkaline phosphatase substrate (Sigma) in 100 mM diethanolamine (pH 8.9). To verify that TG2 remained immobilized during oxidative conditions, one of the GSSGtreated samples was incubated with biotin-CUB7402 (Neomarkers) instead of 5-BP.

TG2 Is Reversibly Inactivated by Oxidation-TG2 is known
to be sensitive to oxidative environments and will gradually lose activity upon improper handling and storage. Regulation of activity through oxidation is also likely to be relevant in vivo. This prompted us to investigate the mechanism behind oxidative inactivation of TG2. Using recombinant human TG2, we observed that prolonged handling of the enzyme in the absence of reducing agents resulted in loss of enzymatic activity (Fig.  1A). This inactivation was found to be reversible, as activity could be restored by treatment with DTT. Similar results were observed upon treatment of TG2 with oxidizing agent by using an excess of GSSG over GSH (Fig. 1B). These observations suggest that loss of enzymatic activity is accompanied by cysteine oxidation, leading to disulfide bond formation. Oxidation was also found to influence the conformation of TG2, rendering the enzyme more prone to assume an open conformation (Fig. 1C). Whereas WT TG2 migrated in a closed conformation upon incubation with GTP, oxidized TG2 was unable to assume or retain a closed conformation during native PAGE. However, treatment with DTT did partially restore this ability (Fig. 1C).
Identification of a Cysteine Triad Forming Two Disulfide Bonds upon Oxidation of TG2-TG2 has no reported disulfide bonds in its native state. Disulfide bonds appear to be formed, however, upon oxidative inactivation of the enzyme. To identify cysteine residues susceptible to oxidation, TG2 was incubated with various ratios of GSH and GSSG, followed by alkylation of all free cysteine residues with IAM (ϩ57 Da). After separation by nonreducing SDS-PAGE, the protein band was excised, and existing disulfide bonds were reduced with DTT and alkylated with IAA (ϩ58 Da), giving a mass difference of 1 Da between the originally reduced and oxidized cysteine residues. Oxidation was quantified by mass spectrometric analysis of the tryptic digests. In the tryptic digest of WT TG2, 16 of 20 cysteine residues were observed by detecting the corresponding tryptic peptides (supplemental Table 1). Among these, Cys 230 , Cys 370 , and Cys 371 were found to be particularly susceptible to oxidation (Fig. 2). The active-site Cys 277 did not undergo oxidation under these conditions ( Fig. 2 and supplemental Fig. S1). Cys 370 participated in two disulfide bonds: a bond with either Cys 230 or its neighbor, Cys 371 ( Fig. 3 and supplemental Fig. S2). As Cys 370 was consistently more oxidized than Cys 371 (supplemental Fig. S3), it is likely that there exists an equilibrium between the Cys 230 -Cys 370 and Cys 370 -Cys 371 disulfide bonds. The structural basis for both of the observed disulfide bonds is evident from the crystal structure of the open form of TG2 (15), where the vicinal disulfide bond between Cys 370 and Cys 371 is in fact present, and Cys 230 is within bonding distance of Cys 370 (4.2 Å) (Fig. 4).
We next quantified the percentage of free versus oxidized Cys 370 and Cys 371 upon GSH/GSSG titration. As expected, we observed increased oxidation upon decreasing GSH/GSSG ratios (Fig. 5A). The Cys 230 -Cys 370 disulfide bond was observed to form more readily under less oxidizing conditions (Fig. 5A, gray squares, only Cys 370 oxidized) than the vicinal Cys 370 -Cys 371 disulfide bond (white squares, both Cys 370 and Cys 371 oxidized). On the other hand, the vicinal disulfide bond progressively dominated under more oxidizing conditions.
Enzymatic Activity Correlates Inversely with the Oxidation of Cys 370 and Cys 371 -We next addressed whether the presence of the identified disulfide bridges influences the enzymatic activity of TG2. Peptide substrate and Ca 2ϩ were added to oxidized TG2, and deamidation was quantified at various time points. The level of deamidation was found to correlate with the percentage of reduced Cys 370 and Cys 371 still present in the enzyme upon the addition of substrate, suggesting that both of these cysteine residues must be reduced in order for the enzyme to be active (Fig. 5B). Similar results were obtained for transamidation (supplemental Fig. S4).

Cys 230 Promotes Inactivation of TG2 and Facilitates Formation of the Vicinal Disulfide Bond between Cys 370 and Cys 371 -
To further establish the role of the three cysteine residues, recombinant TG2 mutants C230A, C370A, and C371A were expressed. First, the enzymatic activity and conformational preference of each mutant was tested. Both the k cat and K m of C230A were similar to those of the WT enzyme, suggesting that the C230A mutant does not impair the catalytic cycle of TG2 (Table 1). Moreover, C230A was able to assume the closed conformation like the WT enzyme (Fig. 6A). In contrast, both C370A and C371A mutants were somewhat impaired in different ways. Substrate recognition was affected for both mutants as evidenced by increased K m values. However, although C370A remained an effective catalyst (the k cat /K m was one-third that of the WT enzyme), the

Oxidation of TG2
AUGUST 13, 2010 • VOLUME 285 • NUMBER 33 JOURNAL OF BIOLOGICAL CHEMISTRY 25405 k cat /K m of C371A was only 5% that of the WT enzyme (Table  1). C370A was able to assume a closed conformation upon incubation with GTP, whereas C371A migrated in the open form under identical conditions (Fig. 6A). Additionally, the presence of reducing agents increased the ability of the WT enzyme to assume a closed conformation, whereas this had no influence on C370A (Fig. 6B).
Treatment of C230A with an excess of GSSG over GSH at 37°C resulted in oxidation and loss of enzymatic activity (data not shown). However, C230A was less susceptible to oxidation (Fig. 6C) and inactivation (data not shown) than the WT enzyme. This difference in inactivation kinetics was even more pronounced when oxidation was performed at 30°C. After a 3-h incubation, WT TG2 was extensively oxidized and almost completely inactivated, whereas C230A retained activity and was minimally oxidized (Fig. 6, D and  E). Additionally, the enzymatic activity of WT TG2 could be recovered by incubation with DTT prior to substrate addition, whereas this made little difference for C230A (Fig. 6, D  and E). This suggests that Cys 230 facilitates formation of the vicinal disulfide bond between Cys 370 and Cys 371 and thereby inactivation of TG2. This role for Cys 230 is supported by direct observation of the Cys 230 -Cys 370 disulfide bond formed in the C371A mutant (supplemental Fig. S5). Despite lacking the ability to form the abovementioned disulfide bridges, C370A was inactivated upon oxidation with GSSG (data not shown). Moreover, the closed form of C370A was more susceptible to opening by oxidation by GSSG than the WT enzyme (data not shown). This could be due to glutathionylation of Cys 230 , which was found to occur very rapidly for the C370A mutant (supplemental Fig. S6) but was not observed for the WT enzyme. Steric hindrance due to this modification is likely to affect both activity and conformation. Notably, no oxidation of Cys 371 was observed in the C370A mutant.
Calcium Protects against Oxidation and Inactivation-Hitherto, all oxidation experiments were performed in the absence of Ca 2ϩ . The effect of calcium on oxidation is challenging to address, as TG2 is prone to exert extensive self-crosslinking. To circumvent this problem, Ca 2ϩ titration was performed in the presence of saturating amounts of peptide substrate. In these experiments, increasing amounts of Ca 2ϩ appeared to protect against oxidation (Fig. 7A). As the lack of oxidation could be due to binding of Ca 2ϩ and/or catalytic turnover of the enzyme, Ca 2ϩ titration was also tested in the absence of substrate using the active-site TG2 mutant C277S (Fig. 7B). Again, Ca 2ϩ clearly protected against oxidation with a dramatic change at ϳ1-3 mM Ca 2ϩ , which is close to the K a of Ca 2ϩ (16). The presence of saturating amounts of substrate in the absence of Ca 2ϩ also had a slight protective effect against oxidation (data not shown).
Fibronectin-bound TG2 Is Inactivated by Oxidation-To address whether TG2 associated with fibronectin is also susceptible to oxidation, TG2 was immobilized on fibronectincoated enzyme-linked immunosorbent assay plates, followed by oxidation with GSSG. Enzymatic activity was then assessed by incubation with 5-BP and Ca 2ϩ , followed by streptavidinalkaline phosphatase to detect incorporated 5-BP. Treatment with 1 mM GSSG completely abrogated enzymatic activity, demonstrating that fibronectin-associated TG2 is also susceptible to oxidation (Fig. 8).

DISCUSSION
We have identified a redox-sensitive cysteine triad consisting of Cys 230 , Cys 370 , and Cys 371 that is involved in oxidative inac-    (19 -21). The observed oxidation was independent of the active-site Cys 277 , indicating the presence of additional redox-sensitive cysteine residues within the enzyme that could not be identified at the time. We have demonstrated that oxidative loss of enzymatic activity correlates with oxidation of three cysteine residues, Cys 230 , Cys 370 , and Cys 371 . These residues are in close proximity in the crystal structures of both the closed and open conformations of TG2 (7,15). Within this triad, Cys 370 participates in two disulfide bonds with either Cys 230 or its neighbor, Cys 371 . The vicinal disulfide bond was observed previously in the open crystal structure (15), but also, in the crystal structure, Cys 230 is observed to be within bonding distance of Cys 370 , providing structural support for our findings. Cys 230 -Cys 370 appears to form under less oxidizing conditions than Cys 370 -Cys 371 , which is progressively formed upon increasingly oxidizing conditions, suggesting the mechanism of inactivation depicted in Fig. 9. Mutation of Cys 230 to alanine rendered the enzyme less susceptible to inactivation by oxidation with reduced formation of Cys 370 -Cys 371 . Thus, the formation of Cys 230 -Cys 370 is observed to facilitate subsequent formation of the vicinal disulfide bond, indicating that Cys 230 is acting as a redox sensor that initiates oxidation and inactivation of TG2. Although vicinal cysteine pairs are found in several transglutaminase isoforms (TG1, TG4, TG5, and TG7), Cys 230 is unique for TG2. It therefore appears that Cys 230 allows an isoform-specific mechanism to regulate transglutaminase activity under oxidizing conditions.
In addition to inhibition of enzymatic activity, oxidation also affects the conformation of TG2, rendering the enzyme more susceptible to assuming an open conformation as resolved by native PAGE. This can be reversed by treatment with DTT, Values are given as the mean of two experiments. D and E, oxidation of Cys 370 and Cys 371 was observed by MALDI-TOF mass spectrometry as a shift in the isotopic envelope of the signal for the alkylated peptide at m/z 1481.8 (alkylated with IAM at each cysteine residue giving a mass shift of 2 ϫ 57 Da). Increasing oxidation is seen as a skewing toward m/z 1483.1, which corresponds to alkylation of both cysteine residues with IAA (ϩ2 ϫ 58 Da). Oxidation is indicated as by asterisks. The WT enzyme (D) was oxidized, whereas the C230A mutant (E) was only slightly affected after a 3-h incubation with 0.24 mM GSH and 2 mM GSSG at 30°C. The activity of the oxidized samples was measured before (ox) and after (ox ϩ DTT) incubation with 10 mM DTT for 10 min at room temperature. Activity is given as percent deamidation after 90 min using 90 M DQ2-␣-II and 0.1 g/l enzyme.
indicating that disulfide bond formation impedes flexibility in the hinge of the enzyme. In line with this, we observed that the C370A mutant, which cannot form the two identified disulfide bonds, migrated in a closed conformation irrespective of the presence of reducing agents, whereas reducing agents were clearly beneficial for the WT enzyme. On the other hand, the C371A mutant migrated only in an open conformation, which might be due to stable Cys 230 -Cys 370 bond formation.
The transglutaminase activity of TG2 is strictly Ca 2ϩ -dependent. It is known that binding of Ca 2ϩ ions induces conformational changes in the enzyme that expose the active site (4,5). The presence of Ca 2ϩ also influences oxidation of TG2, although there are conflicting reports about this in the literature (19,20). We observed that Ca 2ϩ protected against formation of the Cys 230 -Cys 370 and Cys 370 -Cys 371 disulfide bonds. Interestingly, Cys 230 resides within one of the proposed Ca 2ϩbinding sites of TG2 (23). Occupancy of this site by Ca 2ϩ could thus prevent formation of the Cys 230 -Cys 370 bond. Conversely, once this bond is formed, it would impede binding of Ca 2ϩ , resulting in loss of enzymatic activity. Notably, the Cys 370 -Cys 371 bond was found to induce significant changes in the peptide backbone conformation, which might influence overall binding of Ca 2ϩ to TG2 (15).
TG2 is transported across the plasma membrane by an unknown mechanism. It is associated with integrins on the cell surface (24), and it can be found deposited in the extracellular matrix tightly bound to fibronectin (25). How TG2 activity in the extracellular compartment is regulated is unclear, as the presence of Ca 2ϩ and the absence of GTP should allow for constitutive activation. A recent study demonstrated that extracellular TG2 is catalytically inactive during homeostasis and is only transiently activated upon exposure to certain inflammatory stimuli and upon tissue injury (9). A burst in activity upon cell wounding could be detected only immediately after injury and was completely diminished after 12 h. This is in line with a model wherein TG2 is released into the extracellular matrix and remains catalytically active for a short period of time but then becomes silenced through oxidation. Although found to protect against disulfide bond formation in our experiments, Ca 2ϩ has been reported to promote inactivation of TG2 through nitrosylation (26). This was also found to be reversible, although it is not clear whether inactivation derives from tyrosine nitrosylation or nitrosylation of the active-site cysteine (27). Modification of gluten peptides by TG2 is an essential step in the pathogenesis of celiac disease. Based on our results, we hypothesize that if this modification takes place in the extracellular environment, either it must take place soon after release from injured cells before TG2 is inactivated by oxidation, or  Incorporation of 40 M 5-BP by fibronectin-immobilized TG2 in the presence of 10 mM CaCl 2 was measured after incubation with 0 mM GSSG or 1 mM GSSG. TG2 was still bound to fibronectin after GSSG treatment as detected by monoclonal antibody CUB7402. some yet unknown mechanism keeps the enzyme active in an oxidative extracellular environment. Further studies are required to address this issue.