[This corrects the article on p. 100 in vol. 41.].

Energy conservation in chemotrophic anaerobic bacteria.

Protein Radicals in Enzyme Catalysis.

As a normal attribute of aerobic life, structural damage to organic compounds of a wide variety (DNA, proteins, carbohydrates and lipids) may occur as a consequence of oxidative reactions. Oxidative damage inflicted by reactive oxygen species has been called “oxidative stress”. Biological systems contain powerful enzymatic and nonenzymatic antioxidant systems, and oxidative stress denotes a shift in the prooxidant/antioxidant balance in favor of the former. Diverse biological processes such as inflammation, carcinogenesis, ageing, radiation damage and photobiological effects appear to involve reactive oxygen species. This field of research provides new perspectives in biochemical pharmacology, toxicology, radiation biochemistry as well as pathophysiology.

Biochemistry of Oxidative Stress

A novel protein superfamily with over 600 members was discovered by iterative profile searches and analyzed with powerful bioinformatics and information visualization methods. Evidence exists that these proteins generate a radical species by reductive cleavage of S-adenosylmethionine (SAM) through an unusual Fe-S center. The superfamily (named here Radical SAM) provides evidence that radical-based catalysis is important in a number of previously well- studied but unresolved biochemical pathways and reflects an ancient conserved mechanistic approach to difficult chemistries. Radical SAM proteins catalyze diverse reactions, including unusual methylations, isomerization, sulfur insertion, ring formation, anaerobic oxidation and protein radical formation. They function in DNA precursor, vitamin, cofactor, antibiotic and herbicide biosynthesis and in biodegradation pathways. One eukaryotic member is interferon-inducible and is considered a candidate drug target for osteoporosis; another is observed to bind the neuronal Cdk5 activator protein. Five defining members not previously recognized as homologs are lysine 2,3-aminomutase, biotin synthase, lipoic acid synthase and the activating enzymes for pyruvate formate-lyase and anaerobic ribonucleotide reductase. Two functional predictions for unknown proteins are made based on integrating other data types such as motif, domain, operon and biochemical pathway into an organized view of similarity relationships.

/pdf/radical-sam-a-novel-protein-superfamily-linking-unresolved-4shlcnhz3o.pdf

Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods

It was once widely held that nearly all reactions in biology were catalyzed via mechanisms involving paired electron species. Beginning approximately 40 years ago, this paradigm was repeatedly challenged as examples of enzymatic reactions involving organic radical intermediates began to emerge, and it is now well accepted that biochemical reactions often involve organic radicals. Indeed, some of the most intensely studied metalloenzymes, including cytochrome P450, methane monooxygenase, ribonucleotide reductase, and the adenosylcobalamin (B12) enzymes, catalyze reactions employing organic radical intermediates. As a general rule, enzymes utilizing radical mechanisms catalyze reactions that would be difficult or impossible to catalyze by polar mechanisms, most often involving H-atom abstraction from an unactivated C–H bond.

Among the more recent additions to the enzymes that catalyze radical reactions are the radical S-adenosylmethionine (radical SAM) enzymes, which were first classified as a superfamily in 2001.1 These enzymes utilize a [4Fe–4S] cluster and SAM to initiate a diverse set of radical reactions, in most or all cases via generation of a 5′-deoxyadenosyl radical (dAdo•) intermediate. Although 2001 marked the identification of this superfamily largely through bioinformatics, the discovery of iron metalloenzymes utilizing SAM to initiate radical reactions precedes this date by more than a decade. For example, early studies on the activation of pyruvate formate-lyase showed that it involved the generation of a stable protein radical,2 and was stimulated by the presence of iron, SAM, and an “activating component” from the cell extract now known to be the pyruvate-formate lyase activating enzyme (PFL-AE).3 The radical on PFL was ultimately shown to be located on a specific glycine residue,4 and was one of the first stable protein radicals characterized. PFL-AE was ultimately shown to contain a catalytically essential iron–sulfur cluster,5 and to use SAM as an essential component of PFL activation.6 The anaerobic ribonucleotide reductase, similar to PFL, contains a stable glycyl radical that was shown in early work to require an iron–sulfur cluster and SAM for activation.7 Likewise, preliminary investigations on lysine 2,3-aminomutase (LAM) published in 1970 demonstrated activation by ferrous ion and a strict requirement for SAM.8 Like PFL-AE, LAM was ultimately found to contain a catalytically essential iron–sulfur cluster.9 Work in Perry Frey’s lab showed that LAM used the adenosyl moiety of SAM to mediate hydrogen transfer in a manner similar to adenosylcobalamin-dependent rearrangements, implicating radical intermediates.10 Biotin synthase was first reported to require iron and SAM in 1995,11 and was subsequently shown to contain iron–sulfur clusters and to catalyze a radical reaction.12

These four enzyme systems (PFL/PFL-AE, aRNR, LAM, and biotin synthase) provided early indications of a new type of biological cofactor consisting of an iron–sulfur cluster and SAM, which initiate radical reactions using a fundamental new mechanism of catalysis.13 What none of us in the field in the early days probably anticipated, however, was just how ubiquitous these enzymes would turn out to be. The initial report of the superfamily by Sofia et al. identified ∼600 members;1 however, now that number is ∼48 100 members.14 These enzymes are found across the phylogenetic kingdom and catalyze an amazingly diverse set of reactions, the vast majority of which have yet to be characterized.

This Review will begin by summarizing unifying features of radical SAM enzymes, and in subsequent sections delve further into the biochemical, spectroscopic, structural, and mechanistic details for those enzymes that have been characterized. In most cases, these enzymes are grouped by reaction type; however, in two cases (syntheses of modified tetrapyrroles and complex metal cluster cofactors), we have chosen to group together several radical SAM enzymes that catalyze different reaction types but which act together in the same or related metabolic pathways.

Radical S-Adenosylmethionine Enzymes

Pyruvate formate-lyase (formate acetyltransferase; EC 2.3.1.54) of Escherichia coli cells is post-translationally interconverted between inactive and active forms. Conversion of the inactive to the active form is catalyzed by an Fe2+-dependent activating enzyme and requires adenosylmethionine and dihydroflavodoxin. This process is shown here to introduce a paramagnetic moiety into the structure of pyruvate formate-lyase. It displays an EPR signal at g = 2 with a doublet splitting of 1.5 mT and could comprise an organic free radical located on an amino acid residue of the polypeptide chain. Hypophosphite was discovered as a specific reagent that destroys both the enzyme radical and the enzyme activity; it becomes covalently bound to the protein. The enzymatic generation of the radical, which is linked to adenosylmethionine cleavage into 5'-deoxyadenosine and methionine, possibly occurs through an Fe-adenosyl complex. These results suggest a radical mechanism for the catalytic cycle of pyruvate formate-lyase.

http://www.pnas.org/content/81/5/1332.full.pdf

Post-translational activation introduces a free radical into pyruvate formate-lyase

Flavodoxin and ferredoxin become reduced in Escherichia coli cells by oxidoreductase reactions which use pyruvate and NADPH as electron donor substrates. The two enzymes, which are minor proteins of this organism, were measured through the reduced flavodoxin-dependent activation of pyruvate formate-lyase. The NADPH-dependent enzyme, obtained homogeneously through Procion-red affinity chromatography, was identified as the flavoprotein 'component R' described previously by Fujii and Huennekens [J. Biol. Chem. 249, 6745-6753 (1974)]. The pyruvate-dependent enzyme was identified as CoA-acetylating pyruvate:flavodoxin (ferredoxin) oxidoreductase. Its catalytic properties in the forward, reverse, and the 14CO2-pyruvate exchange reaction are reported. The dihydro form of flavodoxin was characterized as the particular species involved in the activation of pyruvate formate-lyase. The activation process still occurs with 70% of maximal efficiency when the ratio [NADPH]/([NADP] + [NADPH]) is fixed at the intracellular 'anabolic reduction charge' value of 0.45, in conjunction with the NADPH-dependent enzyme. The [2Fe-2S] ferredoxin, though being readily used as electron acceptor of both oxidoreductases and having a redox potential similar to flavodoxin, proved incompetent in mediating the activation of pyruvate formate-lyase.

https://febs.onlinelibrary.wiley.com/doi/epdf/10.1111/j.1432-1033.1982.tb06569.x

Routes of flavodoxin and ferredoxin reduction in Escherichia coli. CoA-acylating pyruvate: flavodoxin and NADPH: flavodoxin oxidoreductases participating in the activation of pyruvate formate-lyase.

The reaction pyruvate + CoA ⇌ acetyl-CoA + formate, catalyzed by pyruvate formate-lyase of Escherichia coli, occurs by the succeeding half-reactions (a) E + pyruvate ⇌ E-acetyl + formate; (b) E-acetyl + CoA ⇌ E + acetyl-CoA.



Making use of coupled optical assays, a ‘ping-pong mechanism’ was derived from the complete kinetic investigation of the forward and reverse reactions. The thermodynamic equilibrium constant of the overall reaction was calculated from the kinetic constants to be K= 750 (30°C, pH 8.1), which agrees with chemically determined values.



The intermediate acetyl-enzyme, which had been previously proposed from the [14C]formate-pyruvate exchange, was detected by product-pulse experiments with [2-14C]pyruvate and trapped by acid precipitation. The acetyl group is linked to a sulfhydryl group of the protein.



The value of the equilibrium constant of the first half-reaction is about 50, as directly measured and calculated from the kinetic data. It was concluded that the standard free energy of hydrolysis of acetyl-enzyme is about 1.7 kcal (7.1 kJ) more negative than that of acetyl-CoA.



The intermediate was found to react with dithiothreitol with a second-order rate constant at 30°C and pH 7.6 of 1160 M−1× min−1. It resulted in a half-life of 4 s (or 20 s at 0°C) in the particular buffer which was required for enzyme stabilization.



The enzyme (about 60 U/mg) was prepared by carrying its purified inactive form through the enzyme-II-dependent activation reaction, employing photoreduced flavodoxin along with the effector compounds S-adenosylmethionine and oxamate (as a pyruvate analogue).

Hans P. Blaschkowski

Papers

Post-translational activation introduces a free radical into pyruvate formate-lyase

Routes of flavodoxin and ferredoxin reduction in Escherichia coli. CoA-acylating pyruvate: flavodoxin and NADPH: flavodoxin oxidoreductases participating in the activation of pyruvate formate-lyase.

Pyruvate Formate‐Lyase of Escherichia coli: the Acetyl‐Enzyme Intermediate

Pyruvate formate-lyase (inactive form) and pyruvate formate-lyase activating enzyme of Escherichia coli: Isolation and structural properties

Pyruvate formate-lyase from Escherischia coli and its activation system.