Adenosyl radical: reagent and catalyst in enzyme reactions

Abstract

Adenosine is undoubtedly an ancient biological molecule that is a component of many enzyme cofactors: ATP, FADH, NAD(P)H, and coenzyme A, to name but a few, and, of course, of RNA. Here we present an overview of the role of adenosine in its most reactive form: as an organic radical formed either by homolytic cleavage of adenosylcobalamin (coenzyme B(12), AdoCbl) or by single-electron reduction of S-adenosylmethionine (AdoMet) complexed to an iron-sulfur cluster. Although many of the enzymes we discuss are newly discovered, adenosine's role as a radical cofactor most likely arose very early in evolution, before the advent of photosynthesis and the production of molecular oxygen, which rapidly inactivates many radical enzymes. AdoCbl-dependent enzymes appear to be confined to a rather narrow repertoire of rearrangement reactions involving 1,2-hydrogen atom migrations; nevertheless, mechanistic insights gained from studying these enzymes have proved extremely valuable in understanding how enzymes generate and control highly reactive free radical intermediates. In contrast, there has been a recent explosion in the number of radical-AdoMet enzymes discovered that catalyze a remarkably wide range of chemically challenging reactions; here there is much still to learn about their mechanisms. Although all the radical-AdoMet enzymes so far characterized come from anaerobically growing microbes and are very oxygen sensitive, there is tantalizing evidence that some of these enzymes might be active in aerobic organisms including humans.

Figure 1

Generation of adenosyl radicals. Top: radical generation by 1-electron reduction of AdoMet complexed with a [4Fe-4S] cluster; bottom: radical generation by homolysis of the Co-C bond of AdoCbl.

Figure 2

Comparison of the structures of AdoCbl and radical AdoMet enzymes. Left: the structures of biotin synthase ((β/α)₈ complete barrel), glutamate mutase ((β/α)₈ complete barrel) and lysine-2,3-aminomutase ((β/α)₆ 3/4 barrel); the structure of the core barrel domain is highlighted in red and gold. Right: details of the cofactor and substrate interactions for each protein; the 5′-carbon of adenosine is in each case indicated with an arrow.

Figure 3

A minimal mechanism for the 1,2-rearrangements catalyzed by adenosyl radical enzymes, here X may be –OH, –NH₂ or a carbon-containing fragment.

Figure 4

Mechanisms for the carbon skeleton rearrangements catalyzed by the AdoCbl-dependent enzymes 2-methyleneglutarate mutase, methylmalonyl-CoA mutase, isobutyryl-CoA mutase and glutamate mutase.

Figure 5

Mechanism for the 1,2-rearrangement catalyzed by AdoMet-dependent lysine-2,3-aminomutase; a similar mechanism could be drawn for AdoCbl dependent lysine-5,6-aminomutase or ornithine-4,5-aminomutase.

Figure 6

Mechanism for the radical elimination reactions catalyzed by AdoCbl-dependent ethanolamine ammonia lyase and diol dehydratase. The reaction catalyzed by AdoMet dependent DesII, shown below, is thought to occur by a similar mechanism.

Figure 7

Mechanism for the resolution of thymidine dimers in DNA catalyzed by AdoMet-dependent spore photoproduct lyase.

Figure 8

Radicals required for the mechanism of ribonucleotide reductase (RNR) are generated differently by the aerobic (tyrosyl radical), anaerobic (glycyl radical) and AdoCbl-dependent enzymes. However the mechanism for ribonucleotide reduction, involving radical elimination of the 2′-OH group, is the same for all classes of enzyme.

Figure 9

Overview of the reactions catalyzed by glycyl radical enzymes. Each enzyme has a cognate radical AdoMet activase responsible for generating the glycyl radical.

Figure 10

The sulfur insertion reactions catalyzed by biotin synthase, lipoyl synthase, MiaB and RimO.

Figure 11

Mechanism for the sulfur insertion reactions catalyzed by biotin synthase.

Figure 12

The structure of the Fe-Fe hydrogenase cofactor. Two radical AdoMet enzymes, HydE and HydG, are involved in the biosynthesis of the bridging dithiolate moiety.

Figure 13

The radical oxidative decarboxylation reaction catalyzed by HemN during the anaerobic biosynthesis of heme.

Figure 14

The oxidation of the active site serine in sulphatase to formylglycine catalyzed by AtsB under anaerobic conditions.

Figure 15

The natural products shown contain methyl groups (indicated in bold) introduced at non-nucleophilic sites that are derived from MeCbl in radical AdoMet-dependent reactions. The proposed mechanism for the methylation reaction catalyzed by Fom3 is shown below.

Figure 16

Radical AdoMet enzymes in thiamine pyrophosphate biosynthesis. Top: the structure of thiamine pyrophosphate; atoms derived from AIR are shown in blue, those derived from tyrosine are shown in red. Middle: mechanism for the formation of dehydroglycine catalyzed by ThiH. Bottom: the reaction catalyzed by ThiC; the color coding indicates where the atoms of the substrate appear in the product, as established by labeling studies.

Figure 17

The conversion of GTP to precursor Z catalyzed by MoaA and MoaC in a radical AdoMet-dependent reaction. Precursor Z is then converted to molybdopterin through subsequent reactions catalyzed by MoaB, MoaD and MoaE.

Figure 18

The biosynthetic pathway for wybutosine in tRNA^phe. The addition of the two carbon fragment shown in bold in the second step of the pathway is catalyzed by TWY1; the identity of the precursor to these carbons, “X”, is currently unknown.