Identification, purification, and characterization of iminodiacetate oxidase from the EDTA-degrading bacterium BNC1

Abstract

Microbial degradation of synthetic chelating agents, such as EDTA and nitrilotriacetate (NTA), may help immobilizing radionuclides and heavy metals in the environment. The EDTA- and NTA-degrading bacterium BNC1 uses EDTA monooxygenase to oxidize NTA to iminodiacetate (IDA) and EDTA to ethylenediaminediacetate (EDDA). IDA- and EDDA-degrading enzymes have not been purified and characterized to date. In this report, an IDA oxidase was purified to apparent homogeneity from strain BNC1 by using a combination of eight purification steps. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein band of 40 kDa, and by using size exclusion chromatography, we estimated the native enzyme to be a homodimer. Flavin adenine dinucleotide was determined as its prosthetic group. The purified enzyme oxidized IDA to glycine and glyoxylate with the consumption of O2. The temperature and pH optima for IDA oxidation were 35 degrees C and 8, respectively. The apparent Km for IDA was 4.0 mM with a kcat of 5.3 s(-1). When the N-terminal amino acid sequence was determined, it matched exactly with that encoded by a previously sequenced hypothetical oxidase gene of BNC1. The gene was expressed in Escherichia coli, and the gene product as a C-terminal fusion with a His tag was purified by a one-step nickel affinity chromatography. The purified fusion protein had essentially the same enzymatic activity and properties as the native IDA oxidase. IDA oxidase also oxidized EDDA to ethylenediamine and glyoxylate. Thus, IDA oxidase is likely the second enzyme in both NTA and EDTA degradation pathways in strain BNC1.

FIG. 1

SDS-PAGE of purified IDA oxidase. Lanes 1 and 3, low-range molecular mass standards (Bio-Rad); lane 2, 0.5 μg of IDA oxidase purified from BNC1; lane 4, 3 μg of C-fusion His tag IdaA purified from E. coli.

FIG. 2

Mass spectra of the ED-dansyl adduct (A) and the end product-dansyl adduct (B).

FIG. 3

Oxygen consumption by IDA oxidase. After 6 min of the reaction, catalase was added as indicated by the arrow.

FIG. 4

The conserved FAD-binding domains (boxed) of IDA oxidase and related enzymes DAAO, HcnC, NoxB, and GloX.

FIG. 5

NTA (A) and EDTA (B) degradation in strain BNC1. EmoA is EDTA monooxygenase.