This is the first report that EDTA-Mo has been identified, purified, and characterized. When supplied with FMNH2
, the enzyme appears to break the N-C bond in EDTA, NTA, and DTPA, with one oxygen atom appearing in glyoxylate. The enzyme was able to degrade EDTA, NTA, and DTPA with a variety of metal cations. With cell extracts, the highest rate was found when Mg2+
was added. The purified enzyme showed similar preferences for MgEDTA2−
and free EDTA. Kinetic analyses indicate that the enzyme has a higher affinity for MgEDTA2−
. This finding coincides with Mg2+
being the most abundant intracellular cation in several tested microorganisms (12
), with MgEDTA2−
the likely form of EDTA in the cytoplasm of BNC1.
HPLC, GC-MS, and colorimetric analyses showed that EDTA was oxidized to glyoxylate and ED3A, and NTA was oxidized to glyoxylate and iminodiacetate by EDTA-Mo (Fig. ). Upon examination of the structures of NTA and ED3A, we wondered whether EDTA-Mo could also degrade ED3A to EDDA. However, GC-MS analysis did not detect any trace of either symmetric or unsymmetric EDDA. We are currently cloning the gene encoding EDTA-Mo by using a DNA probe generated from the N-terminal amino acid sequence. Successful production of a functional EDTA-Mo in an expression host will allow us to confirm the identities of enzyme end products from EDTA and NTA as well as to accumulate enough end products from DTPA for identification. Characterization of the gene sequence will reveal the relationships among related enzymes, especially between EDTA-Mo and NTA monooxygenase (NTA-Mo).
Proposed conversion of EDTA to ED3A and glyoxylate by EDTA-Mo.
The stoichiometry of EDTA oxidation by EDTA-Mo was studied. One molecule of FMN was reduced to one FMNH2 by NAD(P)H:FMN oxidoreductase at the expense of one NADH. Then one FMNH2 reacted with one O2 chemically to generate one H2O2. When catalase was added, one O2 was produced from two H2O2. When EDTA-Mo was also present in the reaction mixture, the enzyme consumed one O2 and one FMNH2 to oxidized EDTA and produced one glyoxylate. It took 12 min to complete O2 consumption in the absence of EDTA-Mo but only 4 min in the presence of EDTA-Mo. Ninety-five percent of FMNH2 generated by NAD(P)H:FMN oxidoreductase was used by EDTA-Mo to oxidize EDTA, and only 5% reacted with O2 to produce H2O2. Although the concentrations of EDTA and ED3A were not determined, the conversion of EDTA to ED3A was determined by GC-MS analysis. On the basis of these data, the reaction catalyzed by EDTA-Mo is proposed (Fig. ).
When the kinetic parameters for EDTA oxidation were determined, excess NAD(P)H:FMN oxidoreductase was added in the reaction mixture. Under such conditions, a substantial amount of H2O2 was produced. Catalase was added to the reaction mixture to prevent the buildup of H2O2. This practice provided sufficient amount of FMNH2 to EDTA-Mo. When O2 consumption was studied, excess EDTA-Mo was added to effectively utilize FMNH2 so that the formation of H2O2 was minimized. Because of FMNH2 is highly reactive with O2, we could not use O2 as the limiting factor in the reaction mixture and thus could not determine its kinetic parameters.
There are similarities and differences between EDTA-Mo of BNC1 and NTA-Mo (formerly component A) of C. heintzii
). Both enzymes require FMNH2
as cosubstrates, and both are single polypeptides of about 50 kDa. EDTA-Mo oxidizes EDTA to ED3A and glyoxylate, and oxidizes NTA to iminodiacetate and glyoxylate. NTA-Mo oxidizes NTA to iminodiacetate and glyoxylate (37
). NTA-Mo degrades only specific metal-NTA complexes (37
), while EDTA-Mo degrades EDTA, NTA, and DTPA in the absence or presence of metal cations. NTA-Mo does not degrade EDTA or DTPA.
Flavin-dependent monooxygenases are ubiquitous. FAD and FMN are normally prosthetic groups, not cosubstrates, and they are reduced by the monooxygenases themselves with NADH or NADPH as the reductant (8
). EDTA-Mo uses FMNH2
directly. Since the enzyme does not appear to contain any chromophores and does not require any specific transitional metal cofactors, the activated oxygen species is likely a C(4a)-flavin hydroperoxide as proposed for other flavin-dependent monooxygenases (15
). Thus, FMNH2
acts as both reductant and prosthetic group for EDTA-Mo. An endogenous FMN reductase supplied EDTA-Mo with FMNH2
. Since the reductase was replaced by two other FMN reductases, it is unlikely that there is any direct protein-protein interactions between the reductase and oxygenase. These data suggest that EDTA-Mo belongs to a small group of monooxygenases that utilize FMNH2
as both reductant and prosthetic group. Bacterial luciferase of P. fischeri
was the first FMNH2
-utilizing monooxygenase studied (36
). Recently, pristinamycin IIA synthase of Streptomyces pristinaespiralis
), NTA-Mo of C. heintzii
), and two monooxygenases involved in desulfurization of dibenzothiophene by Rhodococcus
sp. strain IGTS8 (11
) have been characterized and shown to use FMNH2
as the cosubstrate. EDTA-Mo appears to be the sixth member of this group. These FMNH2
-dependent monooxygenases appear to attack carbon-nitrogen, carbon-sulfur, carbon-carbon, or carbon-oxygen double bonds.