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

Poly(ethylene terephthalate) (PET) is used extensively worldwide in plastic products, and its accumulation in the environment has become a global concern. Because the ability to enzymatically degrade PET has been thought to be limited to a few fungal species, biodegradation is not yet a viable remediation or recycling strategy. By screening natural microbial communities exposed to PET in the environment, we isolated a novel bacterium, Ideonella sakaiensis 201-F6, that is able to use PET as its major energy and carbon source. When grown on PET, this strain produces two enzymes capable of hydrolyzing PET and the reaction intermediate, mono(2-hydroxyethyl) terephthalic acid. Both enzymes are required to enzymatically convert PET efficiently into its two environmentally benign monomers, terephthalic acid and ethylene glycol.

http://science.sciencemag.org/content/sci/351/6278/1196.full.pdf

A Bacterium That Degrades and Assimilates Poly(ethylene Terephthalate)

Some bacteria think plastic is fantastic Bacteria isolated from outside a bottle-recycling facility can break down and metabolize plastic. The proliferation of plastics in consumer products, from bottles to clothing, has resulted in the release of countless tons of plastics into the environment. Yoshida et al. show how the biodegradation of plastics by specialized bacteria could be a viable bioremediation strategy (see the Perspective by Bornscheuer). The new species, Ideonella sakaiensis, breaks down the plastic by using two enzymes to hydrolyze PET and a primary reaction intermediate, eventually yielding basic building blocks for growth. Science, this issue p. 1196; see also p. 1154 Two specialized enzymes from a newly isolated bacterium break down plastic into its simplest building blocks. [Also see Perspective by Bornscheuer] Poly(ethylene terephthalate) (PET) is used extensively worldwide in plastic products, and its accumulation in the environment has become a global concern. Because the ability to enzymatically degrade PET has been thought to be limited to a few fungal species, biodegradation is not yet a viable remediation or recycling strategy. By screening natural microbial communities exposed to PET in the environment, we isolated a novel bacterium, Ideonella sakaiensis 201-F6, that is able to use PET as its major energy and carbon source. When grown on PET, this strain produces two enzymes capable of hydrolyzing PET and the reaction intermediate, mono(2-hydroxyethyl) terephthalic acid. Both enzymes are required to enzymatically convert PET efficiently into its two environmentally benign monomers, terephthalic acid and ethylene glycol.

A bacterium that degrades and assimilates poly (ethylene terephthalate) and its enzymes involved in the degradation

A central issue in nitric oxide (NO) research is to understand how NO can act in some settings as a servoregulator and in others as a cytotoxin. To answer this, we have sought a molecular basis for the differential regulation of the two known types of NO synthase (NOS). Constitutive NOS's in endothelium and neurons are activated by agonist-induced elevation of Ca2+ and resultant binding of calmodulin (CaM). In contrast, NOS in macrophages does not require added Ca2+ or CaM, but is regulated instead by transcription. We show here that macrophage NOS contains, as a tightly bound subunit, a molecule with the immunologic reactivity, high performance liquid chromatography retention time, tryptic map, partial amino acid sequence, and exact molecular mass of CaM. In contrast to most CaM-dependent enzymes, macrophage NOS binds CaM tightly without a requirement for elevated Ca2+. This may explain why NOS that is independent of Ca2+ and elevated CaM appears to be activated simply by being synthesized.

/pdf/calmodulin-is-a-subunit-of-nitric-oxide-synthase-from-31l05pr1mm.pdf

Calmodulin is a subunit of nitric oxide synthase from macrophages.

Phytochemicals: The Chemical Components of Plants H.L. Brielmann Jr., W.N. Setzer, P.B. Kaufman, A. Kirakosyan, and L.J. Cseke How and Why These Compounds Are Synthesized by Plants L.J. Cseke, C.R. Lu, A. Kornfeld, P.B. Kaufman, and A. Kirakosyan Regulation of Metabolite Synthesis in Plants L.J. Cseke and P. B. Kaufman Plant Natural Products in the Rhizosphere Bhinu, V.S., K. Narasimhan, and S. Swarup Molecular Biology of Plant Natural Products Sheela Reuben, L.J. Cseke, Bhinu, V.S., K. Narasimhan, M. Jeyakumar and S. Swarup The Study of Natural Product Biosynthesis in the Pregenomics and Genomics Eras F. Chen, L.J. Cseke, H. Lin, A. Kirakosyan, J.S. Yuan, and P. Kaufman Plant Biotechnology for the Production of Natural Products A. Kirakosyan Traditional, Analytical, and Preparative Separations of Natural Products L.J. Cseke, W.N. Setzer, B. Vogler, A. Kirakosyan, and P.B. Kaufman Characterization of Natural Products B. Vogler and W.N. Setzer Bioassays for Activity W.N. Setzer and B. Vogler Modes of Action at Target Sites S. Warber, E.M. Seymour, P. Kaufman, A. Kirakosyan, and L.J. Cseke The Uses of Plant Natural Products by Humans and Risks Associated with Their Use P.B. Kaufman, A. Kirakosyan, M. McKenzie, P. Dayanandan, J.E. Hoyt, and C. Li The Synergy Principle at Work with Plants, Pathogens, Insects, Herbivores, and Humans K. Spelman, J.A. Duke and M.J. Bogenschutz-Godwin Plant Conservation M.J. Bogenschutz-Godwin, J.A. Duke, M. McKenzie, and P.B. Kaufman Relationship Between People and Plants S. Warber and K. Irvine Appendix:Information Retrieval on Natural Products in Plants Indexes: Chemicals, Plant Species and Common Names, and Subject

Hitoshi Fujisawa

Papers

Protocatechuate 3,4-Dioxygenase I. CRYSTALLIZATION AND CHARACTERIZATION

Protocatechuate 3,4-Dioxygenase II. ELECTRON SPIN RESONANCE AND SPECTRAL STUDIES ON INTERACTION OF SUBSTRATES AND ENZYME

Protocatechuate 3,4-Dioxygenase III. AN OXYGENATED FORM OF ENZYME AS REACTION INTERMEDIATE

Studies on the Mechanism of Action Pyrocatechase by Electron Spin Resonance Spectroscopy

Oxygenated form of protocatechuate 3,4-dioxygenase, a non-heme iron-containing dioxygenase, as reaction intermediate.