Choice of microbial host for the naphthalene dioxygenase bioconversion

The use of whole cell biotransformations for single and multistep enzyme conversions is gaining widespread application. In this study the naphthalene dioxygenasenah A gene was transferred intoPseudomonas aeruginosa PAC 1R,Escherichia coli JM107 andPseudomonas putida PpG 277. The effect of ethanol on these genetically engineered Gram-negative bacteria was studied by measurement of enzyme activity, stability and cell integrity. Ethanol has been used in biotransformations as a co-substrate carbon source for co-factor recycling and as a co-solvent increasing dissolved substrate and product levels. Ethanol increased the dissolved substrate (naphthalene) concentration slightly and dissolved product ((+)-cis-(1R, 2S)-dihydroxy-1,2-dihydronaphthalene) by approximately 30% at 4% (w/v) ethanol. BothP. aeruginosa PAC 1R andP. putida PpG 277 showed decreased activity with increasing ethanol concentration whilstE. coli enzyme activity increased with increasing ethanol concentration being comparable to that when glucose was used as a carbon source. This project highlighted the many factors involved in the selection of microbial hosts for whole cell biotransformation processes.     

Biodegradation of 4-chlorobiphenyl by using induced cells and cell extract of Burkholderia xenovorans

This study aimed to evaluate the efficiency of Burkholderia xenovorans LB400 cells and their cell extract to remediate 4-chlorobiphenyl (4-CB). The bacterium previously induced with 4-CB was able to degrade up to 98% of initial 50 mg L^?1 of 4-CB from mineral medium within 96 h of incubation. The degradation of 4-CB occurred through the formation of meta-cleavage product 2-hydroxy-6-oxo-6phenylhexa-2,4-dienoic acid (HOPDA), as revealed through enzymatic assay of 2,3-dihydroxybiphenyl 1,2-dioxygenase (2,3-DHBD). A derivative of 1,2-benzenedicarboxylic acid was observed as one of the major intermediate metabolites of 4-CB degradation. Time course production of 2,3-DHBD during growth corresponds with the degradation pattern of 4-CB by the bacterium. In vitro degradation of 4-CB using cell extract of B. xenovorans showed complete degradation of initial 25 mg L^?1 of 4-CB within 6 h of incubation. To the best of the authors' knowledge, this is the first report in which in vitro degradation of 4-CB using cell extract of Burkholderia xenovorans is presented.     

The apple gene responsible for columnar tree shape reduces the abundance of biologically active gibberellin

Ectopic expression of the apple 2-oxoglutarate-dependent dioxygenase (DOX, 2ODD) gene, designated MdDOX-Co, is thought to cause the columnar shape of apple trees. However, the mechanism underlying the formation of such a unique tree shape remains unclear. To solve this problem, we demonstrated that Arabidopsis thaliana overexpressing MdDOX-Co contained reduced levels of biologically active gibberellin (GA) compared with wild type. In summary: (i) with biochemical approaches, the gene product MdDOX-Co was shown to metabolize active GA A₄ (GA₄) to GA₅₈ (12-OH-GA₄) in vitro. MdDOX-Co also metabolized its precursors GA₁₂ and GA₉ to GA₁₁₁ (12-OH-GA₁₂) and GA₇₀ (12-OH-GA₉), respectively; (ii) Of the three 12-OH-GAs, GA₅₈ was still active physiologically, but not GA₇₀ or GA₁₁₁; (iii) Arabidopsis MdDOX-Co OE transformants converted exogenously applied deuterium-labeled (d₂)-GA₁₂ to d₂-GA₁₁₁ but not to d₂-GA₅₈, whereas transformants converted applied d₂-GA₉ to d₂-GA₅₈; (iv) GA₁₁₁ is converted poorly to GA₇₀ by GA 20-oxidases in vitro when GA₁₂ is efficiently metabolized to GA₉; (v) no GA₅₈ was detected endogenously in MdDOX-Co OE transformants. Overall, we conclude that 12-hydroxylation of GA₁₂ by MdDOX-Co prevents the biosynthesis of biologically active GAs in planta, resulting in columnar phenotypes.     

Degradation of Phenanthrene by Mutant Naphthalene-Degrading Pseudomonas putida Strains

Five naphthalene- and salicylate-utilizing Pseudomonas putida strains cultivated for a long time on phenanthrene produced mutants capable of growing on this substrate and 1-hydroxy-2-naphthoate as the sole sources of carbon and energy. The mutants catabolize phenanthrene with the formation of 1-hydroxy-2-naphthoate, 2-hydroxy-1-naphthoate, salicylate, and catechol. The latter products are further metabolized by the meta- and ortho-cleavage pathways. In all five mutants, naphthalene and phenanthrene are utilized with the involvement of plasmid-born genes. The acquired ability of naphthalene-degrading strains to grow on phenanthrene is explained by the fact that the inducible character of the synthesis of naphthalene dioxygenase, the key enzyme of naphthalene and phenanthrene degradation, becomes constitutive.     

Ethylene biosynthesis: The role of 1-aminocyclopropane-1-carboxylate (ACC) oxidase, and its possible evolutionary origin

Recent developments in our knowledge of the biochemistry of 1-aminocyclopropane-1-carboxylate (ACC) oxidase, the enzyme responsible for the final stage in the biosynthesis of ethylene, are reviewed. Particular reference is made to the role of carbon dioxide as an essential cofactor, the activity of ACC oxidase in the plant cell, the enzyme catalytic centre, and the role of ACC oxidase in the evolutionary development of ethylene biosynthesis in plants. Evidence is marshalled to support a proposal that the membrane requirement for ACC oxidase that is observed in vivo is attributable to a need for a charged plasma membrane to maintain ascorbate in the reduced state for an ACC oxidase located in the apoplast. It is argued on biochemical grounds that the acquisition of the ACC oxidase was the crucial evolutionary step in the development by seed plants of an ethylene biosynthesis pathway that could easily be regulated, and that signalled the plant's response to stress and pathogen attack.     

Cytochrome P450–redox partner fusion enzymes

The cytochromes P450 (P450s) are a broad class of heme b-containing mono-oxygenase enzymes. The vast majority of P450s catalyse reductive scission of molecular oxygen using electrons usually derived from coenzymes (NADH and NADPH) and delivered from redox partner proteins. Evolutionary advantages may be gained by fusion of one or more redox partners to the P450 enzyme in terms of e.g. catalytic efficiency. This route was taken by the well characterized flavocytochrome P450_BM3 system (CYP102A1) from Bacillus megaterium, in which soluble P450 and cytochrome P450 reductase enzymes are covalently linked to produce a highly efficient electron transport system for oxygenation of fatty acids and related molecules. However, genome analysis and ongoing enzyme characterization has revealed that there are a number of other novel classes of P450–redox partner fusion enzymes distributed widely in prokaryotes and eukaryotes. This review examines our current state of knowledge of the diversity of these fusion proteins and explores their structural composition and evolutionary origins.     

Purification and properties of ferredoxinBPH, a component of biphenyl 2,3-dioxygenase of Pseudomonas sp strain LB400

The ferredoxin component (ferredoxin_BPH) of biphenyl 2,3-dioxygenase was purified to homogeneity from crude cell extract of Pseudomonas sp strain LB400 using ion exchange, hydrophobic interaction and gel filtration column chromatography. The protein was a monomer with a molecular weight of 15000 and contained 2 gram-atoms each of iron and acid-labile sulfur. Ultraviolet-visible absorbance spectroscopy showed peaks at 325 nm and 460 nm with a broad shoulder around 575 nm. The spectrum was partially bleached in the visible region upon reduction by reductase_BPH with NADPH as the source of electrons. Electron paramagnetic resonance spectrometry showed no signals for the oxidized protein. Upon reduction with sodium dithionite, signals with g_x = 1.82, g_y = 1.92 and g_z = 2.02 were detected. These results indicate that the protein contains a Rieske-type (2Fe-2S) iron-sulfur center. Ferredoxin_BPH was required for the oxidation of biphenyl by the terminal oxygenase component of the enzyme and is probably involved in the transfer of reducing equivalents from reductase_BPH to the terminal oxygenase during catalysis. Received 01 November 1996/ Accepted in revised form 27 May 1997     

Conversion of indene to cis-(1S),(2R)-indandiol by mutants of Pseudomonas putida F1

Two mutation and selection methods were used to isolate mutants of Pseudomonas putida F1 which convert indene to cis-(1S),(2R)-indandiol in a toluene-independent fashion. Using soybean or silicone oil as a second phase to deliver indene to the culture, cis-(1S),(2R)-indandiol, cis-(1R),(2S)-indandiol, 1,2-indenediol (or the keto-hydroxy indan tautomer), and the monooxygenation products 1-indenol and 1-indanone were produced from indene as a function of time. Similarly the enantiomeric excess of the cis-(1S),(2R)-indandiol produced also increased with increasing time. In addition, mutants were isolated which produced cis-(1S),(2R)-indandiol of lower optical purity which corresponded to reduced levels of 1,2-indenediol. These data suggest this toluene dioxygenase produces cis-(1S),(2R)-indandiol of low optical purity and that cis-glycol dehydrogenase plays a role in resolving the two cis-1,2-indandiol enantiomers. Received 15 November 1996/ Accepted in revised form 09 March 1997