ion step follows the decarboxylation, which is consistent with the deuterium isotopic effects observed for thymine 7-hydroxylase which indicate that an irreversible step (or steps) occurs prior to the C-H bond breaking.395 It has also been shown for prolyl 4-hydroxylase that a substrate-derived radical is generated in the reaction, which is consistent with a rebound mechanism.437 It is important to point out that no oxygen intermediate (i.e., bridged superoxo or oxo-ferryl) has been observed for any R-KGdependent enzyme. This warrants future theoretical and experimental study. A detailed molecular mechanism has been proposed for IPNS based on spectroscopic and crystallographic studies.422 Resting IPNS/FeII is also 6C and thus relatively stable toward dioxygen. Substrate ACV binds directly to FeII IPNS through its thiolate group, providing an open coordination position at the FeII. O2 can then react to form an FeIII-superoxo intermediate. This intermediate is suggested422 to perform the first hydrogen-atom abstraction step and close the â-lactam ring, resulting in the formation of the first water molecule and generating an FeIVdO-II intermediate, which completes the second ringclosure process by hydrogen-atom abstraction forming a thiazolidine ring. Previously proposed mechanisms of ACCO involved direct binding of cosubstrate ascorbate to the iron before O2 as part of the oxygen activation process.438,439 The EPR and ESEEM studies of the NO complex of ACCO suggested a quite different molecular mechanism for ACCO.435 An FeIII-superoxo intermediate is proposed. Whether it is preceded by a 6C f 5C process with substrate binding is presently under study.440 This intermediate is thought to initiate a radical process by single hydrogen-atom abstraction or electron-coupled proton transfer (PT)ion or electron-coupled proton transfer (PT) from the bound amino group. The resulting substrate radical may undergo spontaneous conversion into products. The role of cosubstrate ascorbate is proposed to reduce the toxic peroxo byproduct to water. Alternatively, the two-electron reduction of FeIIIsuperoxo by the cosubstrate ascorbate could result in an FeIVdO-II intermediate which initiates the radical reaction.435 4. Rieske-Type Dioxygenases Biochemical Characterization. The Rieske ironsulfur center is a two iron-two sulfur cluster ([2Fe2S]) which has a 2His (on one iron), 2Cys (on the other iron) coordination environment, instead of the 4Cys present in plant ferredoxins. It plays a key role in the electron transport pathway in membranebound cytochrome complexes as well as in some dioxygenases.441 The latter are mainly comprised of two protein components: a reductase containing flavin and a ferredoxin [2Fe-2S], and a terminal oxygenase containing a Rieske [2Fe-2S] cluster and a non-heme iron active site.442 Except for the recently reported alkene monooxygenase that has a binuclear iron site in its terminal oxygenase,10 most of the Rieske-type oxygenases have a mononuclear iron site, which is believed to be the site of dioxygen activation and substrate oxygenation.442,443 The majority of the Rieske-type mononuclear non-heme oxygenases form a family of enzymes which are aromatic-ring-hydroxylating dioxygenases. These catalyze the regioand stereospecific cis-dihydroxylation of an aromatic ring using dioxygen and NAD(P)H (Table 1). Examples include benzene dioxygenase (BDO, EC 1.14.12.3),444 phthalate dioxygenase (PDO, EC 1.14.12.7),445 toluene dioxygenase (EC 1.14.12.11),446 and naphthalene 1,2-dioxygenase (NDO, EC 1.14.12.12),447 which initiate the aerobic degradation of aromatic compounds in the soil bacteria and are targets for bioengineering in bioremediation. This step is the first step in the pathway that ultimately leads to ring cleavage by the intraand extradiol dioxygenases (sections II.B.2 and II.C.1).443 Besides these bacterial dioxygenases, other Rieske-type mononuclear non-heme oxygenases include anthranilate 1,2-dioxygenase (EC 1.14.12.1),448 which deaminates and decarboxylates the substrate to produce catechol; chlorophenylacetate 3,4-dioxygenase (EC 1.14.2.13),449 which converts substrate to catechol with chloride elimination; and 4-methoxybenzoate O-demethylase (putidamonooxin),450 which catalyzes the conversion of 4-methoxybenzoic acid to 4-hydroxybenzoic acid and formaldehyde. The reductase component is usually a monomer (MW ) 12-15 kDa) and utilizes flavin to mediate ET from the two-electron donor NAD(P)H to the oneelectron acceptor [2Fe-2S] cluster and is specific to each terminal oxygenase; other electron donors do not support efficient oxygenation.442 The crystal structure of phthalate dioxygenase reductase is available.451 The terminal oxygenases are large protein aggregates (MW ) 150-200 kDa) containing either multiples of R subunits (BDO R2, PDO R4) or an equimolar combination of R and â subunits (toluene dioxygenase R2â2, NDO R3â3). The R subunits contain a Rieske [2Fe-2S] cluster and a catalytic non-heme FeII center. â subunits do not seem to be involved in the catalytic function (vide infra). Kinetics. Steady-state kinetic studies coupled with various rapid reaction studies of the partial reactions of PDO allowed Ballou et al. to propose a kinetic scheme (Scheme 15).443 On the basis of steady state 278 Chemical Reviews, 2000, Vol. 100, No. 1 Solomon et al.

Geometric and Electronic Structure/Function Correlations in Non-Heme Iron Enzymes

The present invention is generally directed to the evolution of new metabolic pathways and the enhancement of bioprocessing through a process herein termed recursive sequence recombination. Recursive sequence recombination entails performing iterative cycles of recombination and screening or selection to “evolve” individual genes, whole plasmids or viruses, multigene clusters, or even whole genomes. Such techniques do not require the extensive analysis and computation required by conventional methods for metabolic engineering.

Methods and compositions for cellular and metabolic engineering

Summary Members of the genus Burkholderia are versatile organisms that occupy a surprisingly wide range of ecological niches. These bacteria are exploited for biocontrol, bioremediation and plant growth promotion purposes, but safety issues regarding human infections, especially in cystic fibrosis patients, have not been solved. This minireview gives an overview of the taxonomic and ecological diversity of the genus with particular emphasis on strains belonging to the Burkholderia cepacia complex and addresses the important question whether ‘good’ and ‘bad’ strains are actually the same. The genus Burkholderia contains over 30 species, which occupy remarkably diverse ecological niches, ranging from contaminated soils to the respiratory tract of humans. The Burkholderia cepacia complex is ubiquitous in nature and can be found in soil, water (including sea water), the rhizosphere of plants, in humans and various animal species and in the hospital environment. Burkholderia cepacia complex isolates have been exploited for various purposes, including biological control of plant pathogens, bioremediation of recalcitrant xenobiotics and plant growth promotion. Unfortunately, some Burkholderia species have been involved in human infections, and safety issues regarding these human infections are hampering the wide-spread biotechnological applications. The purpose of this minireview is to give an overview of the remarkable diversity of the genus Burkholderia and to shed some light on the important question whether or not clinical and environmental B. cepacia complex isolates are the same. A phylogenetic tree based on 16S rRNA gene sequences, showing the positions of all of the Burkholderia species and representatives of related genera, is shown in Fig. 1.

Diversity and significance of Burkholderia species occupying diverse ecological niches

Enzyme immobilization has attracted continuous attention in the fields of fine chemistry, biomedicine, and biosensor. The performance of immobilized enzyme largely depends on the structure of supports. Nanostructured supports are believed to be able to retain the catalytic activity as well as ensure the immobilization efficiency of enzyme to a high extent. Electrospinning provides a simple and versatile method to fabricate nanofibrous supports. Compared with other nanostructured supports (e.g. mesoporous silica, nanoparticles), nanofibrous supports show many advantages for their high porosity and interconnectivity. This review mainly discusses the recent advances in using nanofibers as hosts for enzyme immobilization by two different methods, surface attachment and encapsulation. Surface attachment refers to physical adsorption or covalent attachment of enzymes on pristine or modified nanofibrous supports, and encapsulation means electrospinning a mixture of enzyme and polymer. We make a detailed comparison between these two immobilization approaches and highlight their distinct characteristics. The prospective applications of enzyme immobilized electrospun nanofibers in the development of biosensors, biofuel cells and biocatalysts are also discussed.

Enzyme immobilization on electrospun polymer nanofibers: An overview

Biodegradation potential of the genus Rhodococcus.

Genetic analysis of a Pseudomonas locus encoding a pathway for biphenyl/polychlorinated biphenyl degradation.

The biphenyl/polychlorinated biphenyl-degradation locus (bph) of Pseudomonas sp. LB400 encodes four additional metabolic enzymes.

Metabolism of 21 chlorobiphenyls by the enzymes of the upper biphenyl catabolic pathway encoded by the bph locus of Pseudomonas sp. strain LB400 was investigated by using recombinant strains harboring gene cassettes containing bphABC or bphABCD. The enzymes of the upper pathway were generally able to metabolize mono- and dichlorinated biphenyls but only partially transform most trichlorinated congeners investigated: 14 of 15 mono- and dichlorinated and 2 of 6 trichlorinated congeners were converted into benzoates. All mono- and at least 8 of 12 dichlorinated congeners were attacked by the bphA-encoded biphenyl dioxygenase virtually exclusively at ortho and meta carbons. This enzyme exhibited a high degree of selectivity for the aromatic ring to be attacked, with the order of ring preference being non- > ortho- > meta- > para-substituted for mono- and dichlorinated congeners. The influence of the chlorine substitution pattern of the metabolized ring on benzoate formation resembled its influence on the reactivity of initial dioxygenation, suggesting that the rate of benzoate formation may frequently be determined by the rate of initial attack. The absorption spectra of phenylhexadienoates formed correlated with the presence or absence of a chlorine substituent at an ortho position.

Conversion of chlorobiphenyls into phenylhexadienoates and benzoates by the enzymes of the upper pathway for polychlorobiphenyl degradation encoded by the bph locus of Pseudomonas sp. strain LB400.

Burkholderia sp. strain LB400 is one of the most potent aerobic polychlorobiphenyl (PCB)-degrading microorganisms that have been characterized. Its PCB-dioxygenating activity originates predominantly or exclusively from the biphenyl dioxygenase encoded by its bph gene cluster. Analysis of the dioxygenation products of several di- to pentachlorinated biphenyls formed by this enzyme revealed a complex dependence of the regiospecificity and the yield of dioxygenation on the substitution patterns of both the oxidized and the nonoxidized rings. No dioxygenolytic attack involving chlorinated meta or para carbons was observed. Therefore, the ability of the enzyme to hydroxylate chlorinated carbons appears to be limited to the ortho position. However, it is not limited to monochlorinated rings, as evidenced by dioxygenation of the 2,4-disubstituted ring at carbons 2 and 3. This site of attack is strikingly different from that of the 2,5-dichlorinated ring, which has been shown to be dihydroxylated at positions 3 and 4 (J. D. Haddock, J. R. Horton, and D. T. Gibson, J. Bacteriol. 177:20–26, 1995). These results demonstrate that a second substituent of ortho-chlorinated rings crucially influences the site of dioxygenation at this ring and thereby determines whether or not the initial chlorobiphenyl oxidation product is further metabolized through the bph-encoded pathway. The 2,4-dichlorinated ring can alternatively be attacked at carbons 5 and 6. The preferred site crucially depends on the substitution pattern of the other ring. The formation of more than a single dioxygenation product was found predominantly with congeners that contain two chlorinated rings, both of which are similarly prone to dioxygenation or one is substituted only at carbon 3.

Regiospecificity of Dioxygenation of Di- to Pentachlorobiphenyls and Their Degradation to Chlorobenzoates by the bph-Encoded Catabolic Pathway of Burkholderia sp. Strain LB400

Summary

The degradation of polychlorobiphenyls (PCBs) by diverse bacteria, including Burkholderia sp. LB400, is incomplete with a concomitant accumulation of metabolic intermediates. In this study, the toxicity of diverse (chloro)biphenyls and of their biotransformation into the first two metabolic intermediates of the biphenyl pathway, were determined for the model bacterium Escherichia coli. Recombinant E. coli strains expressing different subsets of bph genes of strain LB400 accumulated metabolic intermediates from (chloro)biphenyls. During biotransformation of these compounds into metabolic intermediates, the viability and metabolic kinetics were determined. The toxicity of biotransformation of (chloro)biphenyls into different metabolic intermediates of (chloro)biphenyls varied. Dihydrodiols and dihydroxybiphenyls are very toxic metabolites for bacteria even after short incubation times, affecting the cell viability much more than (chloro)biphenyls. When bacteria transformed 2-CB into dihydrodiol or dihydroxybiphenyl, a great decrease of intact cells and abundant cell lysis was observed by transmission electronic microscopy. Cell viability of Burkholderia sp. LB400 and of E. coli exposed directly to 2,3-dihydroxybiphenyl decreased also drastically. The toxicity of metabolites generated during oxidation of PCBs may partly explain the recalcitrance to biodegradation of these pollutants. Conversion of less toxic compounds into products with increased toxicity resembles the bioactivation of xenobiotics in higher organisms.

Bernd Hofer

Papers

Genetic analysis of a Pseudomonas locus encoding a pathway for biphenyl/polychlorinated biphenyl degradation.

The biphenyl/polychlorinated biphenyl-degradation locus (bph) of Pseudomonas sp. LB400 encodes four additional metabolic enzymes.

Conversion of chlorobiphenyls into phenylhexadienoates and benzoates by the enzymes of the upper pathway for polychlorobiphenyl degradation encoded by the bph locus of Pseudomonas sp. strain LB400.

Regiospecificity of Dioxygenation of Di- to Pentachlorobiphenyls and Their Degradation to Chlorobenzoates by the bph-Encoded Catabolic Pathway of Burkholderia sp. Strain LB400

From PCBs to highly toxic metabolites by the biphenyl pathway