Directed evolution of aniline dioxygenase for enhanced bioremediation of aromatic amines

The objective of this study was to enhance the activity of aniline dioxygenase (AtdA), a multi-component Rieske non-heme iron dioxygenase enzyme isolated from Acinetobacter sp. strain YAA, so as to create an enhanced biocatalyst for the bioremediation of aromatic amines. Previously, the mutation V205A was found to widen the substrate specificity of AtdA to accept 2-isopropylaniline (2IPA) for which the wild-type enzyme has no activity (Ang EL, Obbard JP, Zhao HM, FEBS J, 274:928–939, 2007). Using mutant V205A as the parent and applying one round of saturation mutagenesis followed by a round of random mutagenesis, the activity of the final mutant, 3-R21, was increased by 8.9-, 98.0-, and 2.0-fold for aniline, 2,4-dimethylaniline (24DMA), and 2-isopropylaniline (2IPA), respectively, over the mutant V205A. In particular, the activity of the mutant 3-R21 for 24DMA, which is a carcinogenic aromatic amine pollutant, was increased by 3.5-fold over the wild-type AtdA, while the AN activity was restored to the wild-type level, thus yielding a mutant aniline dioxygenase with enhanced activity and capable of hydroxylating a wider range of aromatic amines than the wild type. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Assembly of non-natural electron transfer conduits in the cytochrome P450 system: a critical assessment and update of artificial redox constructs amenable to exploitation in biotechnological areas

The high plasticity of the active-site cavity of cytochromes P450, permitting reactivity toward a vast array of compounds, makes these enzymes attractive targets for biotechnological application. Escalating attention in this area is driven by remarkable progress in the rational design by DNA shuffling of self-sufficient, multi-domain P450/electron donor constructs simplifying the composition of biocatalytic systems. Moreover, versatile approaches were undertaken to supersede the well-established, NAD(P)H-steered proteinaceous redox chains by cost-effective alternative electron transfer conduits constituted of organometallic mediators or photoactivatable redox triggers. Electrochemical techniques have proven particularly useful: employing different types of carbon- and metal-based electrodes for the fabrication of biosensors, the continuing challenge was to optimize the conductive properties of these devices by creating biocompatible interfaces for transferring electrons between sensor surfaces and redox proteins. The present review provides a critical update of the most significant breakthroughs in innovative manipulation of the redox machinery, giving an impulse to exploitation of P450s in fields such as the production of fine chemicals, drug processing, medicinal diagnostics and remediation of biotopes contaminated with harmful environmental pollutants.     

Depurinating naphthalene–DNA adducts in mouse skin related to cancer initiation

Naphthalene has been shown to be a weak carcinogen in rats. To investigate its mechanism of metabolic activation and cancer initiation, mice were topically treated with naphthalene or one of its metabolites, 1-naphthol, 1,2-dihydrodiolnaphthalene (1,2-DDN), 1,2-dihydroxynaphthalene (1,2-DHN), and 1,2-naphthoquinone (1,2-NQ). After 4 h, the mice were sacrificed, the treated skin was excised, and the depurinating and stable DNA adducts were analyzed. The depurinating adducts were identified and quantified by ultraperformance liquid chromatography/tandem mass spectrometry, whereas the stable adducts were quantified by ³²P-postlabeling. For comparison, the stable adducts formed when a mixture of the four deoxyribonucleoside monophosphates was treated with 1,2-NQ or enzyme-activated naphthalene were also analyzed. The depurinating adducts 1,2-DHN-1-N3Ade and 1,2-DHN-1-N7Gua arise from reaction of 1,2-NQ with DNA. Similarly, the major stable adducts appear to derive from the 1,2-NQ. The depurinating DNA adducts are, in general, the most abundant. Therefore, naphthalene undergoes metabolic activation to the electrophilic ortho-quinone, 1,2-NQ, which reacts with DNA to form depurinating adducts. This is the same mechanism as other weak carcinogens, such as the natural and synthetic estrogens, and benzene.     

Characterization of novel 2-oxoglutarate dependent dioxygenases converting l-proline to cis-4-hydroxy-l-proline

Hydroxyprolines are valuable chiral building blocks for organic synthesis of pharmaceuticals. Several microorganisms producing l-proline trans-4- and cis-3-hydroxylase were discovered and these enzymes were applied to the industrial production of trans-4- and cis-3-hydroxy-l-proline, respectively. Meanwhile, other hydroxyproline isomers, cis-4- and trans-3-hydroxy-l-proline, were not easily available because the corresponding hydroxylase have not been discovered. Herein we report novel l-proline cis-4-hydroxylases converting free l-proline to cis-4-hydroxy-l-proline. Two genes encoding uncharacterized proteins from Mesorhizobium loti and Sinorhizobium meliloti were cloned and overexpressed in Escherichia coli, respectively. The functions of purified proteins were investigated in detail, and consequently we detected l-proline cis-4-hydroxylase activity in both proteins. Likewise l-proline trans-4-, cis-3-hydroxylase and prolyl hydroxylase, these enzymes belonged to a 2-oxoglutarate dependent dioxygenase family and required a non-heme ferrous ion. Although their reaction mechanisms were similar to other hydroxylases, the amino acid sequence homology was not observed (less than 40%).     

新型白化型除草剂靶标酶对羟苯基丙酮酸双加氧酶及其耐性转基因植物研究进展*

对羟苯基丙酮酸双加氧酶(ρ-hydroxyphenylpyruvate dioxygenase,HPPD;EC 1.13.11.27)催化生物体内对羟苯基丙酮酸与O2作用形成尿黑酸的反应,是植物体中质体醌和生育酚生物合成途径的关键酶。当其活性受到抑制时,植物体中作为类胡萝卜素生物合成途径中最终电子受体和光合链电子传递体的质体醌的生物合成受阻,进而导致类胡萝卜素合成减少,光合链电子传递受阻,致使植物体出现白化症状。目前已经开发了多种以HPPD为靶标的除草剂,该类除草剂及抗除草剂转基因植物研究具有广阔的前景。对这一新型白化型除草剂靶标酶以及耐该类除草剂转基因植物的研究进展作了简要综述。     

Endogenous generation of sulfur dioxide in rat tissues

While sulfur dioxide (SO₂) has been previously known for its toxicological effects, it is now known to be produced endogenously in mammals from sulfur-containing amino acid l-cysteine. l-cysteine is catalyzed by cysteine dioxygenase (CDO) to l-cysteinesulfinate, which converts to β-sulfinylpyruvate through transamination by aspartate aminotransferase (AAT), and finally spontaneously decomposes to pyruvate and SO₂. The present study explored endogenous SO₂ production, and AAT and CDO distribution in different rat tissue. SO₂ content was highest in stomach, followed by tissues in the right ventricle, left ventricle, cerebral gray matter, pancreas, lung, cerebral white matter, renal medulla, spleen, renal cortex and liver. AAT activity and AAT1 mRNA expression were highest in the left ventricle, while AAT1 protein expression was highest in the right ventricle. AAT2 and CDO mRNA expressions were both highest in liver tissue. AAT2 protein expression was highest in the renal medulla, but CDO protein expression was highest in liver tissue. In all tissues, AAT1 and AAT2 were mainly distributed in the cytoplasm rather than the nucleus. These observed differences among tissues endogenously generating SO₂ and associated enzymes are important in implicating the discovery of SO₂ as a novel endogenous signaling molecule.     

An overview of epigenetics and chemoprevention

It is now appreciated that both genetic alteration, e.g. mutations, and aberrant epigenetic changes, e.g. DNA methylation, cause cancer. Epigenetic dysregulation is potentially reversible which makes it attractive as targets for cancer prevention. Synthetic drugs targeting enzymes, e.g. DNA methyltransferase and histone deacetylase, that regulate epigenetic patterns are active in clinical settings. In addition, dietary factors have been suggested to have potential to reverse aberrant epigenetic patterns. Uncovering the human epigenome can lead us to better understand the dynamics of DNA methylation in disease progression which can further assist in cancer prevention.     

Phylogenetic analysis and evolutionary studies of plant carotenoid cleavage dioxygenase gene

The oxidative breakdown of carotenoid evidences the formation of apocarotenoids through carotenoid cleavage dioxygenases (CCDs). Numerous CCDs and apocarotenoids have been identified and characterized in plants. Using available sequence data, a study was performed to investigate the phylogenetic relationship among CCD genes and to statistically estimate the sequence conservation and functional divergence. In total, 77 genes were identified from 39 species belonging to 21 families. Our result of phylogenetic analysis indicated the existence of well-conserved subfamilies. Moreover, comparative genomic analysis showed that the gene structures of the CCDs were highly conserved across some different lineage species. Through functional divergence analysis, a substantial divergence was found between CCD subfamilies. In addition, examination of the site-specific profile revealed the critical amino acid residues accounting for functional divergence. This study mainly focused on the evolution of CCD genes and their functional divergence which may deliver an initial step for further experimental verifications.     

Effect of yeast extract on fluoranthene degradation and aromatic ring dioxygenase expressing bacterial community structure of a fluoranthene degrading bacterial consortium

Fluoranthene (Fla) is a high molecular weight polycyclic aromatic hydrocarbon that exerts hazardous effects on living organisms. An efficient Fla degrading bacterial consortium LP was enriched from an oil contaminated soil sample, with and without yeast extract as a supplement. Objective of the present study was to see if there was any differential effect of yeast extract addition on Fla degradation potential and aromatic ring dioxygenase expressing bacteria (ARDB) of the enrichments. Primary enrichment of the soil sample was carried out in minimal salt medium (MSM) added with 500 mg l⁻¹ Fla and 0.05% yeast extract (YMSM). Secondary, tertiary and subsequent enrichments were prepared in YMSM and MSM after every sixteen days of incubation. Fla was efficiently degraded by YMSM enriched culture than MSM enriched culture. However, when MSM enrichment was incubated longer instead of further subculturings, it also degraded Fla efficiently. All three enrichments exhibited growth of bacterial colonies on Fla sprayed minimal agar plates however only YMSM enrichment showed clear zone forming bacterial colonies. A positive effect was observed of yeast extract on ARDB population of LP consortium. To our limited knowledge this is first time that effect of yeast extract on ARDB population was studied.