Thiolate ligation of the active site iron(II) of isopenicillin N synthase derives from substrate rather than endogenous cysteine: spectroscopic studies of site-specific Cys .fwdarw. Ser mutated enzymes

Orville, Allen M.; Chen, Victor; Kriauciunas, Aidas; Harpel, Mark R.; Fox, Brian G.; Münck, Eckard; Lipscomb, John D.

doi:10.1021/bi00134a010

Cited by 103 publications

(97 citation statements)

References 47 publications

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“…The Fe(IV)=O species resulting from the O-O bond cleavage then becomes a reagent for the second ring-closing reaction. It is postulated that it abstracts a hydrogen atom from the β-carbon of the valinyl moiety of ACV 79,87 . This radical intermediate rebounds to the original cysteinyl sulfur ligand rather than the hydroxyl ligand to form the thiazolidine ring of penicillin N and provide the final electron required to form the second water from the oxidase reaction.…”

Section: Oxidase Enzymes In the 2-his+asp/glu Familymentioning

confidence: 99%

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Versatility of biological non-heme Fe(II) centers in oxygen activation reactions

2008

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Oxidase and oxygenase enzymes allow the use of relatively unreactive O 2 in biochemical reactions. Many of the mechanistic strategies employed in nature for this key reaction are represented within the 2-His-1-carboxylate facial triad family of non-heme Fe(II) containing enzymes. The open face of the metal coordination sphere opposite the three endogenous ligands participates directly in the reaction chemistry. Here, data from several studies are presented showing that reductive O 2 activation within this family is initiated by substrate (and in some cases co-substrate or cofactor) binding, which then allows coordination of O 2 to the metal. From this starting point, both the O 2 activation process and the reactions with substrates diverge broadly. The reactive species formed in these reactions have been proposed to encompass four oxidation states of iron and all forms of reduced O 2 as well as several of the reactive oxygen species that derive from O-O bond cleavage.Dioxygen serves at least three quite different roles that profoundly impact aerobic life. The most commonly appreciated role is to serve as the terminal electron acceptor in processes such as oxidative phosphorylation that yield the central energy-rich molecules used throughout metabolism. The second, no less important role is to serve as the source for many of the oxygen atoms found in the essential molecules of biological systems such as steroid hormones, aromatic amino acids, neurotransmitters, signalling molecules, and regulatory factors 1 . Also, processes operating on a global scale, such as the recovery of the enormous quantities of carbon sequestered as lignin in plant life or the oxidation of the billions of tons of methane generated by anaerobes before it can enter the atmosphere, also involve oxygen incorporation from O 2 2 ,3 . On a more local scale, biodegradation of both aliphatic and aromatic toxic compounds often begins with the incorporation of oxygen 4 . The third, and least appreciated role played by dioxygen in aerobic organisms involves neither energy conversion nor oxygen incorporation. Rather, some enzymes can convert dioxygen to alternative forms that are, in effect, highly specialized reagents that are used to catalyze the synthesis of important biomolecules. An excellent example of the latter role for O 2 is the biosynthesis of penicillintype antibiotics 5,6 , which is discussed in more detail later in this review.Dioxygen is an attractive reagent for use in a biological system because its high potential reactivity is held in check by its molecular structure. The triplet ground state of O 2 that results from the presence of two unpaired electrons in degenerate molecular orbitals makes the direct reaction with singlet molecules, the spin-paired state of most potential reaction partners, a forbidden process 7 . The central question that has faced chemists and biochemists for the half Competing Interests StatementThe authors declare no competing financial interests. Of course, the answers to this question are of fundam...

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Section: Oxidase Enzymes In the 2-his+asp/glu Familymentioning

confidence: 99%

“…Spectroscopic and crystallographic studies show that substrate, δ-(L-α-aminoadipoyl)-Lcysteinyl-D-valine (ACV, 6) binds to the iron via its cysteinyl sulfur 80,87 . The O 2 analog NO can be bound in an adjacent site suggesting that oxygen and substrate bind at the same time to the iron 88 .…”

Section: Oxidase Enzymes In the 2-his+asp/glu Familymentioning

confidence: 99%

Versatility of biological non-heme Fe(II) centers in oxygen activation reactions

2008

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“…The solid line is a theoretical simulation of oxidized center II from D. desulfuricans desulfoferrodoxin, using the parameters published in Ref. 16. cysteinyl sulfur (30) as detected in center II of desulfoferrodoxin (15).…”

Section: Fig 4 Mö Ssbauer Spectrum Of the As-isolated (A) And Ascormentioning

confidence: 99%

Neelaredoxin, an Iron-binding Protein from the Syphilis Spirochete, Treponema pallidum, Is a Superoxide Reductase

Jovanović

Ascenso

Hazlett

et al. 2000

Journal of Biological Chemistry

101

119

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Treponema pallidum, the causative agent of venereal syphilis, is a microaerophilic obligate pathogen of humans. As it disseminates hematogenously and invades a wide range of tissues, T. pallidum presumably must tolerate substantial oxidative stress. Analysis of the T. pallidum genome indicates that the syphilis spirochete lacks most of the iron-binding proteins present in many other bacterial pathogens, including the oxidative defense enzymes superoxide dismutase, catalase, and peroxidase, but does possess an orthologue (TP0823) for neelaredoxin, an enzyme of hyperthermophilic and sulfate-reducing anaerobes shown to possess superoxide reductase activity. To analyze the potential role of neelaredoxin in treponemal oxidative defense, we examined the biochemical, spectroscopic, and antioxidant properties of recombinant T. pallidum neelaredoxin. Neelaredoxin was shown to be expressed in T. pallidum by reverse transcriptase-polymerase chain reaction and Western blot analysis. Recombinant neelaredoxin is a 26-kDa ␣ 2 homodimer containing, on average, 0.7 iron atoms/subunit. Mö ssbauer and EPR analysis of the purified protein indicates that the iron atom exists as a mononuclear center in a mixture of high spin ferrous and ferric oxidation states. The fully oxidized form, obtained by the addition of K 3 (Fe(CN) 6 ), exhibits an optical spectrum with absorbances at 280, 320, and 656 nm; the last feature is responsible for the protein's blue color, which disappears upon ascorbate reduction. The fully oxidized protein has a A 280 /A 656 ratio of 10.3. Enzymatic studies revealed that T. pallidum neelaredoxin is able to catalyze a redox equilibrium between superoxide and hydrogen peroxide, a result consistent with it being a superoxide reductase. This finding, the first description of a T. pallidum iron-binding protein, indicates that the syphilis spirochete copes with oxidative stress via a primitive mechanism, which, thus far, has not been described in pathogenic bacteria.

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“…We have studied many of these 2H1C enzymes over the years in studies that I have not had space to describe here. These include Allen Orville's study of isopenicillin N-synthetase (74), Amy Rocklin's study of 1-aminocyclopropane-1-carboxylic acid oxidase (75); Aimin Liu's study of (S)-2-hydroxypropylphosphonic acid epoxidase (76); and a twodecade-long study of Rieske dioxygenases initiated by students Matt Wolfe, Daniel Altier, Matt Neibergall, and Sarmistha Chakrabarty and being carried on by Brent Rivard and Melanie Rogers (72,(77)(78)(79). All of these studies started with a collaboration with another laboratory that complemented our interests and techniques.…”

Section: One Ligand Set Many Mechanismsmentioning

confidence: 99%

Life in a Sea of Oxygen

Lipscomb¹

2014

Journal of Biological Chemistry

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Learning to ListenD uring my third week of high school chemistry, our instructor left in the middle of class in obvious pain and passed away shortly thereafter. This sad event could have spelled disaster for a budding career in chemistry, except for the fact that it occurred at Brandywine High School near Wilmington, Delaware, where 2,500 Ph.D. chemists lived in the school district. These included my father, Robert D. Lipscomb, who, like so many of his DuPont Central Research & Development colleagues, was a product of the Roger Adams/Reynold Fuson/Carl "Speed" Marvel/John Bailar, Jr., era at the University of Illinois. Chemistry class was taken over in principle by a substitute, who was an excellent football coach but knew little chemistry. In fact, it became a learning experiment, fed vicariously by a community of nearly unparalleled depth of expertise and, as I came to experience for the first time, an inbred commitment to understanding over learning. Our fathers and mothers taught us every night, and we taught each other in class. We learned to hear how others interpreted the principles of chemistry and to evaluate quickly how this fit into our own framework for understanding. I have used these lessons, both the importance of listening and the commitment to the profession, continuously in the fifty years that have since passed. I have encountered them in one form or another in each of my mentors. My graduate school advisor I. C. "Gunny" Gunsalus said it very concisely: "Science is about 'learning to listen'." Learn to listen to what your collaborators and students say beneath the words and to what experiments tell you beyond your expectations. CommunicationThese early lessons were reinforced in spades during my time at Amherst College as an undergraduate. At the time, Amherst used a core curriculum, so everyone took the same set of classes in the freshman year, and classes drew context from each other. History was focused on Newton's era the same week that Newtonian principles were encountered in physics, and the physics professor proffered his thoughts on how those principles drove history. Learning to write and scrape away a few protective layers to express how you really understood a problem was a pervasive goal in every class, but especially in the venerable English 1 and 2 courses. In those days, it was perfectly acceptable to hold up a trivial attempt at truth by one of us for class ridicule in anticipation of the one glorious day when our composition was read with a modicum of praise. Trial by fire taught us that communication in all forms that conveys what we actually mean lies at the heart of creative advances of all types, including science. I learned many facts in courses at Amherst, nearly all of which have been superseded by the ferocious pace of the advances that have occurred in all fields over recent decades. In contrast, the lessons in critical writing and communication still resonate, and in a real sense, they are the key to any success that I have enjoyed.At Amherst, I was a chemistry major...

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Thiolate ligation of the active site iron(II) of isopenicillin N synthase derives from substrate rather than endogenous cysteine: spectroscopic studies of site-specific Cys .fwdarw. Ser mutated enzymes

Cited by 103 publications

References 47 publications

Versatility of biological non-heme Fe(II) centers in oxygen activation reactions

Versatility of biological non-heme Fe(II) centers in oxygen activation reactions

Neelaredoxin, an Iron-binding Protein from the Syphilis Spirochete, Treponema pallidum, Is a Superoxide Reductase

Life in a Sea of Oxygen

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