Purification and Properties of p-Hydroxybenzoate Hydroxylases from Rhodococcus Strains.

Gram-positive bacteria of the genus Rhodococcus catabolize p-hydroxybenzoate (PHB) through the initial formation of 3,4-dihydroxybenzoate. High levels of p-hydroxybenzoate hydroxylase (PHBH) activity are induced in six different Rhodococcus species when these strains are grown on PHB as sole carbon source. The PHBH enzymes were purified to apparent homogeneity and appeared to be homodimers of about 95 kD with each subunit containing a relatively weakly bound FAD. In contrast to their counterparts from gram-negative microorganisms, the Rhodococcus PHBH enzymes prefer NADH to NADPH as external electron donor. All purified enzymes were inhibited by Cl– and for five of six enzymes more pronounced substrate inhibition was observed in the presence of chloride ions

Biocatalytic potential of p-hydroxybenzoate hydroxylase from Rhodococcus rhodnii 135 and Rhodococcus opacus 557

The biocatalytic potential of the NADH-dependent p-hydroxybenzoate hydroxylases (PHBH) from Rhodococcus rhodnii 135 and Rhodococcus opacus 557 was investigated. Monofluorinated 4-hydroxybenzoates were efficiently hydroxylated, albeit at different rates. 2-Fluoro-4-hydroxybenzoate was a true substrate for PHBH from R. rhodnii 135 but a substrate inhibitor for PHBH from R. opacus 557. Monochlorinated 4-hydroxybenzoates also acted as PHBH substrates, but with these compounds strong uncoupling of hydroxylation (formation of hydrogen peroxide) occurred. PHBH from R. rhodnii 135 preferred the 5'-hydroxylation of 2-chloro-4-hydroxybenzoate but the enzyme from R. opacus 557 favored the formation of 2-chloro-3,4-dihydroxybenzoate. Conversely, PHBH from R. rhodnii 135 regioselectively hydroxylated 2-fluoro-4-hydroxybenzoate to 2-fluoro-3,4-dihydroxybenzoate whereas the enzyme from R. opacus 557 also produced significant amounts of 2-fluoro-4,5-dihydroxybenzoate. At high NADH/substrate ratio, both 2-fluorodihydroxybenzoate products were further converted to 2-fluoro3,4,5-trihydroxybenzoate. PHBH from R. rhodnii 135 and R. opacus 557 preferred the 5'-hydroxylation of 3-chloro-4-hydroxybenzoate. However, conversion of 3-fluoro-4-hydroxybenzoate involved considerable dehalogenation affording nearly equal amounts of 3,4-dihydroxybenzoate and 5-fluoro-3,4-dihydroxybenzoate. At high NADH/substrate ratio, the latter compound was further converted to 3,4,5-trihydroxybenzoate. The results are discussed in relation to the properties of the NADPH-specific PHBH from Pseudomonas fluorescens

Bioluminescent and spectroscopic properties of His-Trp-Tyr triad mutants of obelin and aequorin.

Ca2+-regulated photoproteins are responsible for the bioluminescence of a variety of marine organisms, mostly coelenterates. The photoproteins consist of a single polypeptide chain to which an imidazopyrazinone derivative (2-hydroperoxycoelenterazine) is tightly bound. According to photoprotein spatial structures the side chains of His175, Trp179, and Tyr190 in obelin and His169, Trp173, Tyr184 in aequorin are at distances that allow hydrogen bonding with the peroxide and carbonyl groups of the 2-hydroperoxycoelenterazine ligand. We replaced these amino acids in both photoproteins by residues with different hydrogen bond donor–acceptor capacity. All mutants exhibited luciferase-like bioluminescence activity, hardly present in the wild-type photoproteins, and showed low or no photoprotein activity, except for aeqH169Q (24% of wild-type activity), obeW179Y (23%), obeW179F (67%), obeY190F (14%), and aeqY184F (22%). The results clearly support the supposition made from photoprotein spatial structures that the hydrogen bond network formed by His–Trp–Tyr triad participates in stabilizing the 2-hydroperoxy adduct of coelenterazine. These residues are also essential for the positioning of the 2-hydroperoxycoelenterazine intermediate, light emitting reaction, and for the formation of active photoprotein. In addition, we demonstrate that although the positions of His–Trp–Tyr residues in aequorin and obelin spatial structures are almost identical the substitution effects might be noticeably different

19F NMR metabolomics for the elucidation of microbial degradation pathways of fluorophenols

Of all NMR-observable isotopes 19F is the one most convenient for studies on the biodegradation of environmental pollutants and especially for fast initial metabolic screening of newly isolated organisms. In the past decade we have identified the 19F NMR characteristics of many fluorinated intermediates in the microbial degradation of fluoroaromatics including especially fluorophenols. In the present paper we give an overview of results obtained for the initial steps in the aerobic microbial degradation of fluorophenols, i.e. the aromatic hydroxylation to di-, tri- or even tetrahydroxybenzenes ultimately suitable as substrates for the second step, ring cleavage by dioxygenases. In addition we present new results from studies on the identification of metabolites resulting from reaction steps following aromatic ring cleavage, i.e. resulting from the conversion of fluoromuconates by chloromuconate cycloisomerase. Together the presented data illustrate the potential of the 19F NMR technique for (1) fast initial screening of biodegradative pathways, i.e. for studies on metabolomics in newly isolated microorganisms, and (2) identification of relatively unstable pathway intermediates like fluoromuconolactones and fluoromaleylacetates

Degradation of 3,4-dichloro- and 3,4-difluoroaniline by Pseudomonas fluorescens 26K

3,4-Dichloro- and 3,4-difluoroanilines were degraded by Pseudomonas fluorescens 26-K under aerobic conditions. In the presence of glucose strain degraded 170 mg/L of 3,4-dichloroaniline (3,4-DCA) during 2-3 days. Increasing of toxicant concentration up to 250 mg/L led to degradation of 3,4-DCA during 4 days and its intermediates during 5-7 days. Without cosubstrate and nitrogen source degradation of 3,4-DCA took place too, but more slowly-about 40% of toxicant at initial concentration 75 mg/L was degraded during 15 days. 3,4-Difluoroaniline (3,4-DFA) (initial concentration 170 mg/L) was degraded by Pseudontonas fluorescens 26-K during 5-7 days. The strain was able to completely degrade up to 90 mg/L of 3,4-DFA, without addition of cosubstrate and nitrogen during 15 days. Degradation of fluorinated aniline was accompanied by intensive defluorination. Activity of catechol 2,3-dioxygenase (C2,3DO) (0.230 mumol/min/mg of protein) was found in the culture liquid of the strain, grown with 3,4-DCA and glucose. This fact, as well as, the presence of 3-chloro-4-hydroxyaniline as a metabolite suggested that 3,4-DCA degradation pathway includes dehalogenation and hydroxylation of aromatic ring followed by its subsequent cleaving by C2,3DO. On the contrary, activity of catechol 1,2-dioxygenase (C1,2DO) (0.08 mumol/min/mg of protein) was found in the cell-free extract of biomass grown on 3,4-DFA. 3 -Fluoro-4-hydroxyani line as intermediate was found in this cell-free extract

19F NMR metabolomics for the elucidation of microbial degradation pathways of fluorophenols

In vivo or in situ nuclear magnetic resonance (NMR) offers a powerful tool to study the degradation of xenobiotics by microorganisms. Most studies reported are based on the use of heteronuclei, and experiments with xenobiotics have been limited because specifically labeled xenobiotics are not commercially available, with the exception of 19F and 31P. 1H NMR is, thus, of great interest in this area. To avoid problems caused by the presence of water and intrinsic metabolite signals, some studies were performed using a deuterated medium or specific detection of protons linked to the 13C-15N enriched pattern. We report here the application of in situ 1H NMR, performed directly on culture media, to study the metabolism of heterocyclic compounds. In this review, we show that a common pathway is involved in the biodegradation of morpholine, piperidine, and thiomorpholine by Mycobacterium aurum MO1 and Mycobacterium sp. RP1. In all cases, the first step is the cleavage of the C-N bond, which results in an amino acid. Thiomorpholine is first oxidized to sulfoxide before the opening of the ring. The second step is the deamination of the intermediate amino acid, which leads to the formation of a diacid. We have shown that the cleavage of the C-N bond and the oxidation of thiomorpholine are initiated by reactions involving a cytochrome P450. Journal of Industrial Microbiology & Biotechnology (2001) 26, 2-8