Glutathione-independent maleylacetoacetate isomerase in gram-positive bacteria

Abstract

Nocardia globerula CL1 produced a glutathione-independent maleylacetoacetate isonierase after growth on L-tyrosine. Partial purification of this isomerase demonstrated its independence of low-molecular-weight cofactors such as glutathione. Similar glutathione-independent maleylacetoacetate isomerases were present in three other gram-positive bacteria grown on tyrosine.Maleylacetoacetate, the ring fission product of homogentisate oxidation, has been previously shown to be isomerized to fumarylacetoacetate with a requirement for glutathione in both mammalian (9, 11) and microbial systems (1). In contrast, hydrolysis of maleylacetoacetate has been reported in several strains of Bacillus spp. (3,4). Maleate and acetoacetate are reported to be the products of hydrolysis in these strains of Bacillus spp., but the metabolic fate of maleate is not known. It has been suggested that direct hydrolysis of maleylacetoacetate is a catabolic trait characteristic of bacteria in the genus Bacillus (4).In another paper (7) we provide evidence that gram-positive bacteria isomerized maleylpyruvate to fumarylpyruvate without using glutathione as a cofactor. The approaches used included the demonstration of fumarylpyruvate as an intermediate in gentisate oxidation and the identification of fumaric acid as a product. In addition, a spectrophotometric assay at pH 8.0 allowed us to make a distinction between glutathione-independent isomerization and direct hydrolysis of maleylpyruvate. In this report we use similar methods to show that a number of gram-positive bacteria also isomerize maleylacetoacetate to fumarylacetoacetate, independent of the presence of glutathione. Previous reports on the hydrolysis of maleylpyruvate (4, 5) and maleylacetoacetate (3) by strains of Bacillus spp. have not made this distinction clearly.Organisms were maintained and grown as previously described (7), by using a minimal medium containing 0.05% L-tyrosine supplemented with 0.005% yeast extract. Cell extracts were prepared as previously described (7), by the method of Hughes (8), and protein concentrations were determined by the method of Lowry et al. (10).Maleylacetoacetate was prepared from homogentisate by using a dialyzed extract of tyrosine-grown Moraxella sp.OA3 as described previously for the isolation of maleylpyruvate from gentisate (7). Solutions of fumarylacetoacetate were prepared daily with 0.10 mM maleylacetoacetate in 50 mM Tris buffer (pH 8) and 0.17 mg of protein per ml of ammonium sulfate-precipitated (20 to 60o saturation) and dialyzed cell extracts of L-tyrosine-grown Nocardia globerula CL1. Isomerization was determined by monitoring the shift in the absorbance maximum from 320 toward 340 nm, with UV spectrophotometry. The assay for maleylacetoacetate isomerase was per-* Corresponding author. t Present address: Celanese Research Co., 86 Morris Avenue, Summit, NJ 07901.formed with 0.3 ,umol of maleylacetoacetate and 150 ,umol of Tris buffer (pH 8.0) in a 3.0-ml total volume. When fumarylacetoacetate hydrolase was present at a higher spec...

Metabolic reconstruction of aromatic compounds degradation from the genome of the amazing pollutant-degrading bacteriumCupriavidus necatorJMP134

“…7) includes a homogentisate dioxygenase (HmgA) that opens the aromatic ring of homogentisate producing maleylacetoacetate which, in some bacteria, is directly hydrolyzed, yielding maleate and acetoacetate (Crawford, 1976); nevertheless, in most bacteria it is isomerized into fumarylacetoacetate (Chapman & Dagley, 1962). This isomerization is either catalyzed by a GSH-independent maleylacetoacetate isomerase, as in most Gram-positive bacteria (Hagedorn & Chapman, 1985), or by GSH-dependent enzymes (hmgC gene products), as has been reported in the Gram-negative strains D. acidovorans (Hareland et al, 1975), B. cepacia (Hamzah & Al-Baharna, 2001), and A. evansii KB740 (Mohamed Mel et al, 2002). Finally, fumarylacetoacetate is hydrolyzed by a specific hydrolase (HmgB) forming fumarate and acetoacetate (Fig.…”

Kinetics of the Biotransformation of Maleylacetone and Chlorofluoroacetic Acid by Polymorphic Variants of Human Glutathione Transferase Zeta (hGSTZ1-1)

“…The isomerase activity of GSTZ1-1 with a variety of diketoacids as substrates has been determined with enzymes from different microorganisms ( 23 , 24 , − and rat liver ( , ). Little is known, however, about the kinetics of hGSTZ1-1 with MA as substrate.…”