Purification and characterization of a two-component monooxygenase that hydroxylates nitrilotriacetate from "Chelatobacter" strain ATCC 29600

Abstract

An assay based on the consumption of nitrilotriacetate (NTA) was developed to measure the activity of NTA monooxygenase (NTA-Mo) in cell extracts of "Chelatobacter" strain ATCC 29600 and to purify a functional, NTA-hydroxylating enzyme complex. The complex consisted of two components that easily dissociated during purification and upon dilution. Both components were purified to more than 95% homogeneity, and it was possible to reconstitute the functional, NTA-hydroxylating enzyme complex from pure component A (cA) and component B (cB). cB exhibited NTA-stimulated NADH oxidation but was unable to hydroxylate NTA. It had a native molecular mass of 88 kDa and contained flavin mononucleotide (FMN). cA had a native molecular mass of 99 kDa. No catalytic activity has yet been shown for cA alone. Under unfavorable conditions, NADH oxidation was partly or completely uncoupled from hydroxylation, resulting in the formation of H202.Optimum hydroxylating activity was found to be dependent on the molar ratio of the two components, the absolute concentration of the enzyme complex, and the presence of FMN. Uncoupling of the reaction was favored in the presence of high salt concentrations and in the presence of flavin adenine dinucleotide. The NTA-Mo complex was sensitive to sulfhydryl reagents, but inhibition was reversible by addition of excess dithiothreitol. The Km values for Mg2+-NTA, FMN, and NADH were determined as 0.5 mM, 1.3 ,uM, and 0.35 mM, respectively. Of 26 tested compounds, NTA was the only substrate for NTA-Mo.The complexing agent nitrilotriacetate (NTA) is used for a range of different purposes, and one of its most controversial applications is that as a substitute for sodium triphosphate in laundry detergents (28). Many representatives of both obligately aerobic and facultatively denitrifying microorganisms which can use NTA as a sole source of nitrogen, carbon and energy have been isolated. The majority of such isolates are gram-negative, obligately aerobic rods (1,5,10,14,29) which previously have been identified as Pseudomonas spp. Recently, it has been shown that these isolates belong to a new genus for which the name "Chelatobacter" has been proposed (6).The biochemical pathway for NTA degradation was first investigated in the two virtually identical "Chelatobacter" isolates T23 (1) and ATCC 29600 (9). In both strains, a monooxygenase was reported to be responsible for the oxidative conversion of NTA (24,25). In this paper, the characterization of the NTA-Mo in cell extracts, as well as the purification, reconstitution, and characterization of a functional twocomponent NTA-Mo, is reported. MATERIALS AND METHODSGrowth of the microorganism. "Chelatobacter" strain ATCC 29600 was obtained from the American Type Culture Collection, Rockville, Md., and was maintained on a synthetic medium containing 1 g of NTA liter-' as described previously (5). In order to avoid excretion of large amounts of ammonia, the strain was grown on a mixture of NTA and acetate (1 g of each liter-') for large-scale growth (100 lit...

Functional Analysis of the Small Component of the 4-Hydroxyphenylacetate 3-Monooxygenase of Escherichia coli W: a Prototype of a New Flavin:NAD(P)H Reductase Subfamily

“…The only exceptions were the MsuE and the related SsuE reductases ( Table 2) that lack similarity with any other described reductases (22). The similarities among the reductases correlate with the fact that all of them use the same substrates, i.e., FAD-FMN and NAD(P)H. Although most of the HpaC-like reductase components are colorless proteins, suggesting that flavin is not tightly bound to the enzyme, there are some exceptions, such as the NtaB reductase component of the nitrilotriacetate monooxygenase from Chelatobacter heintzii that shows a typical FMN spectrum although the flavin is not strongly bound to the MsuD (42) P. aeruginosa AF026067 enzyme (40). A similar behavior was also observed with the cBЈ reductase component of the EDTA-monooxygenase (44).…”

Structure of nitrilotriacetate monooxygenase component B fromMycobacterium thermoresistibile

“…An important role in cholesterol metabolism and cell survival could be the reason for the high conservation of these genes. Uncertainties remained in a previous discussion of the natural substrates of NTA-Mo (Uetz et al, 1992). It is possible that the structure of the synthetic NTA and its subsequent degradation products resemble some of the key metabolites in metabolism pathways such as the tricarboxylic acid (TCA) pathway, hence the analogous activity.…”

“…The Pfam database (http://www.sanger.ac.uk/software/ pfam) assigned MthNTA-MoB to the PF01613 family of proteins with the FMN-binding split-barrel motif at an E value of 8.7 Â 10 À40 . This NADH:FMN oxidoreductase or flavin reductase family was first described in the early 1990s and exists in many organisms, primarily Gram-negative bacteria (Uetz et al, 1992;Blanc et al, 1995). These short-chain flavin reductases are involved in a variety of biological reactions and often act in concert with a flavin-dependent monooxygenase which oxidizes through the addition of molecular oxygen (Kirchner et al, 2003;Galá n et al, 2000).…”

“…Like other microorganisms, various mycobacteria can use small organic compounds such as nitrilotriacetate (NTA) as their sole source of nitrogen, carbon and energy, allowing rapid adaptation to varying conditions within the host (Uetz et al, 1992;Bally et al, 1994). Recent reports predicted that M. tuberculosis could subsist on alternative carbon sources during persistence within the human host, specifically during its macrophage infection period, where by nature it needs to endure glucose deficiency and an abundance of fatty acids.…”

Two‐Component Flavin‐dependent Pyrrole‐2‐carboxylate Monooxygenase from Rhodococcus Sp.

“…Another two-component FMN-dependent monooxygenase was isolated from Chelatobacter heintzii converting nitrilotriacetate to iminodiacetate and glyoxylate (Uetz et al, 1992).…”