Methylating agents generate cytotoxic and mutagenic DNA damage. Cells use 3-methyladenine-DNA glycosylases to excise some methylated bases from DNA, and suicidal O(6)-methylguanine-DNA methyltransferases to transfer alkyl groups from other lesions onto a cysteine residue. Here we report that the highly conserved AlkB protein repairs DNA alkylation damage by means of an unprecedented mechanism. AlkB has no detectable nuclease, DNA glycosylase or methyltransferase activity; however, Escherichia coli alkB mutants are defective in processing methylation damage generated in single-stranded DNA. Theoretical protein fold recognition had suggested that AlkB resembles the Fe(ii)- and alpha-ketoglutarate-dependent dioxygenases, which use iron-oxo intermediates to oxidize chemically inert compounds. We show here that purified AlkB repairs the cytotoxic lesions 1-methyladenine and 3-methylcytosine in single- and double-stranded DNA in a reaction that is dependent on oxygen, alpha-ketoglutarate and Fe(ii). The AlkB enzyme couples oxidative decarboxylation of alpha-ketoglutarate to the hydroxylation of these methylated bases in DNA, resulting in direct reversion to the unmodified base and the release of formaldehyde.

The Escherichia coli lipA gene product has been genetically linked to carbon-sulfur bond formation in lipoic acid biosynthesis [Vanden Boom, T. J., Reed, K. E., and Cronan, J. E., Jr. (1991) J. Bacteriol. 173, 6411-6420], although in vitro lipoate biosynthesis with LipA has never been observed. In this study, the lipA gene and a hexahistidine tagged lipA construct (LipA-His) were overexpressed in E. coli as soluble proteins. The proteins were purified as a mixture of monomeric and dimeric species that contain approximately four iron atoms per LipA polypeptide and a similar amount of acid-labile sulfide. Electron paramagnetic resonance and electronic absorbance spectroscopy indicate that the proteins contain a mixture of [3Fe-4S] and [4Fe-4S] cluster states. Reduction with sodium dithionite results in small quantities of an S = 1/2 [4Fe-4S](1+) cluster with the majority of the protein containing a species consistent with an S = 0 [4Fe-4S](2+) cluster. LipA was assayed for lipoate or lipoyl-ACP formation using E. coli lipoate-protein ligase A (LplA) or lipoyl-[acyl-carrier-protein]-protein-N-lipoyltransferase (LipB), respectively, to lipoylate apo-pyruvate dehydrogenase complex (apo-PDC) [Jordan, S. W., and Cronan, J. E. (1997) Methods Enzymol. 279, 176-183]. When sodium dithionite-reduced LipA was incubated with octanoyl-ACP, LipB, apo-PDC, and S-adenosyl methionine (AdoMet), lipoylated PDC was formed. As shown by this assay, octanoic acid is not a substrate for LipA. Confirmation that LipA catalyzes formation of lipoyl groups from octanoyl-ACP was obtained by MALDI mass spectrometry of a recombinant PDC lipoyl-binding domain that had been lipoylated in a LipA reaction. These results provide information about the mechanism of LipA catalysis and place LipA within the family of iron-sulfur proteins that utilize AdoMet for radical-based chemistry.

The reaction of substrate-bound taurine/alpha-ketoglutarate dioxygenase with O2 has been studied using cryogenic continuous-flow spectroscopy. Transient absorption spectra acquired at -38 degrees C show an exponential decay of a 318-nm chromophore with an apparent rate of 1.3 s-1. The observed optical changes and their kinetics are consistent with the profile of an Fe(IV) species detected recently by Mössbauer spectroscopy (Price et al., Biochemistry 2003, 42, 7497-7508). Resonance Raman measurement upon excitation at 363.7 nm reveal at least two oxygen isotope-sensitive vibrations at 821/787 cm-1 and 583/555 cm-1 for 16O and 18O derivatives, respectively. An additional mode is likely to be obscured by an ethylene glycol vibration at 865 cm-1 and/or 1089 cm-1. The 821 cm-1 vibration is assigned to the stretching mode of Fe(IV)=O species on the basis of its frequency and isotopic shift amplitude. The 583 cm-1 band is likely to originate from an Fe-O2 precursor of the Fe(IV)=O species, although its structural details are unclear at present.

No abstract

Pyruvate formate-lyase activating enzyme (PFL-AE) generates the catalytically essential glycyl radical of PFL. It is a member of the so-called "radical-SAM superfamily" of enzymes that use a [4Fe-4S] cluster and S-adenosylmethionine (AdoMet or SAM) to catalyze diverse radical-mediated reactions. Evidence suggests that this class of enzymes operate by common initial steps involving the generation of an AdoMet-derived adenosyl radical intermediate, of which the mechanism remains unresolved. The three-cysteine CX3CX2C cluster-binding motif common to all members of this superfamily suggests a unique Fe site in the [4Fe-4S] cluster, which presumably interacts with AdoMet to effect the reductive cleavage and radical generation. Here we employ a dual-iron-isotope (56Fe/57Fe) approach to demonstrate the existence of a unique Fe site in the [4Fe-4S] cluster of PFL-AE by Mössbauer spectroscopy. Coordination of AdoMet to this unique Fe site was made evident by the observation of a substantial increase in the isomer shift (delta) of the Mössbauer spectrum associated with the unique Fe site: delta = 0.42 mm/s in the absence of AdoMet increases to delta = 0.72 mm/s in the presence of AdoMet. Further, the Mössbauer data show that the binding of AdoMet to the unique Fe site occurs in the [4Fe-4S]2+ state, prior to the injection of the reducing equivalent required for catalysis. This observation indicates that AdoMet coordination is a necessary prerequisite to adenosyl radical generation.

Only a few enzymes are known to utilize stable protein-based radicals as part of their catalytic cycles. 1 Among these are both the aerobic and anaerobic ribonucleotide reductases and pyruvate formate-lyase from Escherichia coli. Aerobic and anaerobic ribonucleotide reductases utilize tyrosyl and glycyl radicals, respectively, to generate a transient active site thiyl radical that effects hydrogen atom abstraction in the initial step of ribonucleotide reduction. 2 Pyruvate formate-lyase (PFL) has been shown to require a stable glycyl radical to effect the rearrangement of pyruvate to formate. 3 The mechanism of generation of these catalytically essential radicals is of considerable interest, but has only been elucidated in any detail for aerobic ribonucleotide reductase. For PFL, an iron-dependent activating enzyme (PFL-AE) is required for activation under reducing conditions in the presence of DTT, via an (S)-adenosylmethionine-dependent hydrogen atom abstraction, to generate a glycyl radical. 4,5 However, the nature of the iron center in PFL-AE has until now remained elusive. We report here the first evidence for the presence of an iron-sulfur cluster in PFL-AE. When a combination of absorption, variable temperature magnetic circular dichroism (VTMCD), EPR, and resonance Raman (RR) spectroscopies is used, anaerobically prepared PFL-AE is shown to contain a mixture of diamagnetic [2Fe-2S] 2+ and [4Fe-4S] 2+ clusters. Since only [4Fe-4S] 2+ clusters remain in dithionite-reduced samples and (S)-adenosylmethionine (SAM) is required to effect reduction to the [4Fe-4S] + state, these results are interpreted in terms of a subunit-bridging [4Fe-4S] 2+,+ cluster in active PFL-AE and suggest that this cluster is involved with generating the 5′-deoxyadenosyl radical from SAM.Purified PFL-AE 6 has a distinct red-brown color and a UVvis absorption spectrum consistent with the presence of an Fe-S cluster (Figure 1a). Analysis of four distinct preparations indicated 1.5 ( 0.1 mol of iron and 1.7 ( 0.2 mol of acidlabile sulfide per mole of enzyme monomer. 7 The specific activity of purified PFL-AE containing 1.5 mol of iron per mole of enzyme was 3800 U/mg, compared to 540 U/mg for enzyme with a low cluster content (<0.2 mol of iron per mole of enzyme), indicating a direct correlation between cluster content and enzyme activity. 8,9 In common with most biological Fe-S centers, reduction with dithionite results in partial bleaching of the visible absorption. However, in contrast to all known types of Fe-S centers which are paramagnetic in at least one oxidation state, neither the as-purified or dithionite-reduced samples contain a paramagnetic Fe-S cluster, as evidenced by parallel and perpendicular X-band EPR and VTMCD studies over the temperature range of 4-50 K. 10 The identity of the diamagnetic Fe-S clusters in these samples was revealed by RR studies in the Fe-S stretching region (Figure 1b). Although the signal-to-noise ratio is poor due to high background fluorescence, the RR spectrum of † Amherst College.

No abstract

Pyruvate formate-lyase activating enzyme utilizes an iron−sulfur cluster and S-adenosylmethionine to generate the catalytically essential glycyl radical on pyruvate formate-lyase. Variable-temperature (4.2−200 K) and variable-field (0.05−8 T) Mössbauer spectroscopy has been used to characterize the iron−sulfur clusters present in anaerobically isolated pyruvate formate-lyase activating enzyme and in the dithionite-reduced form of the enzyme. Detailed analysis of the Mössbauer data indicates that the anaerobically isolated enzyme contains a mixture of Fe−S clusters with the cuboidal [3Fe-4S]+ clusters as the primary cluster form, accounting for 66% of the total iron. Other forms present include [2Fe-2S]2+ (12% of total Fe) and [4Fe-4S]2+ (8% of total iron). Careful examination of Mössbauer spectra recorded at various applied fields reveal a fourth spectral component which is assigned to a linear [3Fe-4S]+ (∼10% of total Fe). Reduction of the as-isolated enzyme by dithionite, interestingly, converts all cluster types into the [4Fe-4S] form with a mixture of 2+ (66% of total iron) and 1+ (12% of total iron) oxidation states. These results are discussed in light of the proposed role for the iron−sulfur cluster in radical generation.