Lysine 2,3-aminomutase Research Articles

Biochemical Characterization of Aminomutases Involved in Salt Tolerance in Methanogenic Archaea

Lysine-2,3-aminomutase (LAM) catalyzes the conversion of L-α-lysine to L-β-lysine, which entails the transfer of an α-amino group to the b-carbon alongside the migration of a β-hydrogen to the α-carbon. This enzyme plays a role in combating salt stress in methanogenic archaea by carrying out the first step in the biosynthesis of Ne-acetyl-β-lysine, a well-studied osmolyte synthesized and accumulated in methanogens. LAM is of special interest as it belongs to the radical S-adenosyl-L-methionine (SAM) superfamily of enzymes which perform diverse and complex chemistry that often produces valuable compounds. Radical SAM enzymes utilize a reduced [4Fe-4S] cluster to initiate the homolytic cleavage of SAM, yielding a highly reactive 5’-deoxyadenosyl radical that then abstracts a hydrogen atom from an otherwise unreactive substrate. Although the bacterial LAM involved in lysine degradation in Clostridium subterminale has been well-characterized, the archaeal LAM involved in osmolyte biosynthesis has never been studied in vitro. Here, we report on the recombinant expression, purification, and enzymatic properties of LAM from Methanococcus maripaludis. The gene encoding LAM from M. maripaludis C7 (MmarC7_0106) was cloned and the his-tagged protein was overexpressed in Escherichia coli. After purification by metal-affinity chromatography under strictly anaerobic conditions, the protein was brown in color, had UV-Vis spectrum characteristics of a [4Fe-4S] cluster that was capable of being reduced to an active state, and had 3.9 mol of iron per monomer. A wide ranged temperature profile study revealed that enzymatic activity was highest at 37°C. The enzyme reaction in the presence of L-lysine, SAM, and dithionite at 37°C had a Kcat = 0.42 s-1 and a Km = 17.8 mM. Finally, alternative substrates for the aminomutation reaction were investigated. There exists another gene annotated as LAM in M. maripaludis C7 due to its close sequence similarity with the bacterial LAM; however, this gene (MmarC7_1783) lacks lysine binding residues and has a 164 amino acid N-terminal extension and as such is referred to as a “long-LAM”. The presence of a “long-LAM” such as MmarC7_1783 in a methanogen is not uncommon as there are several annotated in thermophilic methanogenic species that are known to solely use β-glutamate, another beta-amino acid, as their osmolyte. Given that the only known pathway for the biosynthesis of β-glutamate is through the use of a glutamate-2,3-aminomutase (GAM) and that these thermophilic methanogens do not have an annotated GAM, we proposed that these long-LAMs were in fact GAM which we have now confirmed through heterologous expression studies. Thus, in addition to the biochemical characterization of an archaeal LAM (MmarC7_0106) described above, we have determined that long-LAMs are in fact GAMs required in the biosynthesis of an alternative osmolyte used for survival in high salt concentrations by halotolerant methanogenic archaea.

Why Nature Uses Radical SAM Enzymes so Widely: Electron Nuclear Double Resonance Studies of Lysine 2,3-Aminomutase Show the 5'-dAdo• "Free Radical" Is Never Free.

Lysine 2,3-aminomutase (LAM) is a radical S-adenosyl-L-methionine (SAM) enzyme and, like other members of this superfamily, LAM utilizes radical-generating machinery comprising SAM anchored to the unique Fe of a [4Fe-4S] cluster via a classical five-membered N,O chelate ring. Catalysis is initiated by reductive cleavage of the SAM S-C5' bond, which creates the highly reactive 5'-deoxyadenosyl radical (5'-dAdo•), the same radical generated by homolytic Co-C bond cleavage in B12 radical enzymes. The SAM surrogate S-3',4'-anhydroadenosyl-L-methionine (anSAM) can replace SAM as a cofactor in the isomerization of L-α-lysine to L-β-lysine by LAM, via the stable allylic anhydroadenosyl radical (anAdo•). Here electron nuclear double resonance (ENDOR) spectroscopy of the anAdo• radical in the presence of (13)C, (2)H, and (15)N-labeled lysine completes the picture of how the active site of LAM from Clostridium subterminale SB4 "tames" the 5'-dAdo• radical, preventing it from carrying out harmful side reactions: this "free radical" in LAM is never free. The low steric demands of the radical-generating [4Fe-4S]/SAM construct allow the substrate target to bind adjacent to the S-C5' bond, thereby enabling the 5'-dAdo• radical created by cleavage of this bond to react with its partners by undergoing small motions, ∼0.6 Å toward the target and ∼1.5 Å overall, that are controlled by tight van der Waals contact with its partners. We suggest that the accessibility to substrate and ready control of the reactive C5' radical, with "van der Waals control" of small motions throughout the catalytic cycle, is common within the radical SAM enzyme superfamily and is a major reason why these enzymes are the preferred means of initiating radical reactions in nature.

Travels with Carbon-Centered Radicals. 5′-Deoxyadenosine and 5′-Deoxyadenosine-5′-yl in Radical Enzymology

As a graduate student under Professor R. H. Abeles, I began my journey with 5'-deoxyadenosine, studying the coenzyme B12 (adenosylcobalamin)-dependent dioldehydrase (DDH). I proved that suicide inactivation of dioldehydrase by glycolaldehyde proceeded with irreversible cleavage of adenosylcobalamin to 5'-deoxyadenosine. I further showed that suicide inactivation by [2-(3)H]glycolaldehyde produced 5'-deoxy[(3)H]adenosine, the first demonstration of hydrogen transfer to adenosyl-C5' of adenosylcobalamin. The tritium kinetic isotope effect (T)k was 15, which correlated well with the measurement (D)k = 12 for transformation of [1-(2)H]propane-1,2-diol to [2-(2)H]propionaldehyde by DDH. After establishing my own research program, I returned to the glycolaldehyde inactivation of DDH, showing by EPR that suicide inactivation produced glycolaldehyde-2-yl. In retrospect, suicide inactivation involved scission of adenosylcobalamin to 5'-deoxyadenosine-5'-yl, which abstracted a hydrogen from glycolaldehyde. Captodative-stabilized glycolaldehyde-2-yl could not react further, leading to suicide inactivation. In 1986, my colleagues and I took up the problem of the mechanism by which lysine 2,3-aminomutase (LAM) catalyzes S-adenosylmethionine (SAM) and pyridoxal-5'-phosphate (PLP)-dependent interconversion of l-lysine and l-β-lysine. Because the reaction followed the pattern of adenosylcobalamin-dependent rearrangements, I postulated that SAM might be an evolutionary predecessor to adenosylcobalamin. Testing this hypothesis, we traced hydrogen transfer from lysine through the adenosyl-C5' of SAM to β-lysine. Thus, the 5'-deoxyadenosyl of SAM mediated hydrogen transfer by LAM exactly as in adenosylcobalamin mediated hydrogen transfer in B12-dependent isomerizations. The mechanism postulated that SAM cleaves to form 5'-deoxyadenosine-5'-yl followed by abstraction of C3(H) from PLP-α-lysine aldimine to form PLP-α-lysine-3-yl. PLP-α-lysine-3-yl isomerizes to pyridoxal-β-lysine-2-yl, and a hydrogen abstraction from 5'-deoxyadenosine regenerates 5'-deoxyadenosine-5'-yl and releases β-lysine. Of four radicals in the postulated mechanism, three have been characterized by EPR spectroscopy as kinetically competent intermediates. The analysis of the role of iron allowed researchers to elucidate the mechanism by which SAM is cleaved to 5'-deoxyadenosine-5'-yl. LAM contains one [4Fe-4S] cluster ligated by three cysteine residues. As shown by ENDOR spectroscopy and X-ray crystallography, the fourth ligand to the cluster is SAM, through the methionyl carboxylate and amino groups. Inner sphere electron transfer within the [4Fe-4S](1+)-SAM complex leads to [4Fe-4S](2+)-Met and 5'-deoxyadenosine-5'-yl. The iron-binding motif in LAM, CxxxCxxC, found by other groups in four other SAM-dependent enzymes, is the founding motif for the radical SAM superfamily. These enzymes number in the tens of thousands and are responsible for highly diverse and chemically difficult transformations in the biosphere. Available information supports the hypothesis that this superfamily provides the chemical context from which the much more structurally complex adenosylcobalamin evolved.

Kinetic and Spectroscopic Evidence of Negative Cooperativity in the Action of Lysine 2,3-Aminomutase

Lysine 2,3-aminomutase (LAM) catalyzes the interconversion of L-lysine and L-β-lysine, a component of a number of antibiotics. The reaction requires the cofactors S-adenosyl-L-methionine (SAM), pyridoxal-5'-phosphate (PLP), and a [4Fe-4S] cluster. LAM is a founding member of the radical SAM superfamily of enzymes. LAM is highly specific for L-lysine and will not accept most other amino acids as substrates. L-alanine and L-2-aminobutyrate at 0.2 M react as substrates for LAM at, respectively, 5 × 10(-6) and 8 × 10(-5) times the rate with saturating L-lysine. Saturating ethylamine accelerates the L-alanine reaction 70-fold, and saturating methylamine accelerates the L-2-aminobutyrate reaction 47-fold. The primary amines binding at the active site of LAM with L-alanine or L-2-aminobutyrate simulate L-lysine. The steady-state kinetics of the reaction of L-alanine + ethylamine displays negative cooperativity with respect to L-alanine. The second-order rate constant for production of β-alanine in the reaction of L-alanine and saturating ethylamine is 0.040 M(-1) s(-1), which is 2 × 10(-5) times the value of k(cat)/K(m) for the reaction of L-lysine. When L-lysine is at a concentration 1/16th of K(m), the lysyl-free radical intermediate is hardly detectable by EPR; however, the addition of L-alanine at high concentration (0.2 M) enhances free radical formation, and the addition of ethylamine further enhances radical formation. These facts complement the kinetic observations and support negative cooperativity in the reaction of L-alanine as a substrate for LAM. Present results and independent evidence support negative cooperativity in the reaction of L-lysine as well.

Binding Energy in the One-Electron Reductive Cleavage of S-Adenosylmethionine in Lysine 2,3-Aminomutase, a Radical SAM Enzyme

The common step in the actions of members of the radical SAM superfamily of enzymes is the one-electron reductive cleavage of S-adenosyl-l-methionine (SAM) into methionine and the 5'-deoxyadenosyl radical. The source of the electron is the [4Fe-4S]1+ cluster characterizing the radical SAM superfamily, to which SAM is directly ligated through its methionyl carboxylate and amino groups. The energetics of the reductive cleavage of SAM is an outstanding question in the actions of radical SAM enzymes. The energetics is here reported for the action of lysine 2,3-aminomutase (LAM), which catalyzes the interconversion of l-lysine and l-beta-lysine. From earlier work, the reduction potential of the [4Fe-4S]2+/1+ cluster in LAM is -0.43 V with SAM bound to the cluster (Hinckley, G. T., and Frey, P. A. (2006) Biochemistry 45, 3219-3225), 1.4 V higher than the reported value for trialkylsulfonium ions in solution. The midpoint reduction potential upon binding l-lysine has been estimated to be -0.6 V from the values of midpoint potentials measured with SAM bound to the cluster and l-alanine in place of l-lysine, with S-adenosyl-l-homocysteine (SAH) bound to the cluster in the presence of l-lysine, and with SAH bound to the cluster in the presence of l-alanine or of l-alanine and ethylamine in place of l-lysine. The reduction potential for SAM has been estimated to be -0.99 V from the measured value for S-3',4'-anhydroadenosyl-l-methionine. The reduction potential for the [4Fe-4S] cluster is lowered 0.17 V by the binding of lysine to LAM, and the binding of SAM to the [4Fe-4S] cluster in LAM elevates its reduction potential by 0.81 V. Thus, the binding of l-lysine to LAM contributes 4 kcal mol-1, and the binding of SAM to the [4Fe-4S] cluster in LAM contributes 19 kcal mol-1 toward lowering the barrier for reductive cleavage of SAM from 32 kcal mol-1 in solution to 9 kcal mol-1 at the active site of LAM.

Glutamate 2,3-aminomutase: A new member of the radical SAM superfamily of enzymes

A gene eam in Clostridium difficile encodes a protein that is homologous to lysine 2,3-aminomutase (LAM) in many other species but does not have the lysyl-binding residues Asp293 and Asp330 in LAM from Clostridium subterminale SB4. The C. difficile protein has Lys and Asn, respectively, in the sequence positions of the essential Asp residues in LAM. The C. difficile gene has been cloned into an E. coli expression vector, expressed in E. coli, and the protein purified and characterized. The recombinant protein displays excellent activity as a glutamate 2,3-aminomutase and no activity toward l-lysine. The PLP-, iron-, and sulfide-content and ultraviolet/visible spectrum are similar to LAM, and the enzyme requires SAM and dithionite as activators, as does LAM. Freeze-quench EPR experiments in the presence of l-glutamate reveal a glutamate-based free radical in the steady state of the reaction. A number of other bacterial genomes include genes encoding proteins homologous to the glutamate 2,3-aminomutase from C. difficile, and four of these proteins display the activity of glutamate 2,3-aminomutase when produced in E. coli. All of the homologous proteins have the cysteine motif CSMY CRH C corresponding to the motif CxxxCxxC characteristic of radical SAM enzymes. It is concluded that glutamate 2,3-aminomutase from C. difficile is a representative of a family found in a number of bacteria. It is likely that the β-glutamate found in a few bacterial and archeal species as an osmolyte arises from the action of glutamate 2,3-aminomutase.

Enantiomeric Free Radicals and Enzymatic Control of Stereochemistry in a Radical Mechanism: The Case of Lysine 2,3-Aminomutases

The product of yjeK in Escherichia coli is a homologue of lysine 2,3-aminomutase (LAM) from Clostridium subterminale SB4, and both enzymes catalyze the isomerization of (S)- but not (R)-alpha-lysine by radical mechanisms. The turnover number for LAM from E. coli is 5.0 min(-1), 0.1% of the value for clostridial LAM. The reaction of E. coli LAM with (S)-alpha-[3,3,4,4,5,5,6,6-(2)H8]lysine proceeds with a kinetic isotope effect (kH/kD) of 1.4, suggesting that hydrogen transfer is not rate-limiting. The product of the E. coli enzyme is (R)-beta-lysine, the enantiomer of the clostridial product. Beta-lysine-related radicals are observed in the reactions of both enzymes by electron paramagnetic resonance (EPR). The radical in the reaction of clostridial LAM has the (S)-configuration, whereas that in the reaction of E. coli LAM has the (R)-configuration. Moreover, the conformations of the beta-lysine-related radicals at the active sites of E. coli and clostridial LAM are different. The nuclear hyperfine splitting between the C3 hydrogen and the unpaired electron at C2 shows the dihedral angle to be 6 degrees, unlike the value of 77 degrees reported for the analogous radical bound to the clostridial enzyme. Reaction of (S)-4-thialysine produces a substrate-related radical in the steady state of E. coli LAM, as in the action of the clostridial enzyme. While (S)-beta-lysine is not a substrate for E. coli LAM, it undergoes hydrogen abstraction to form an (S)-beta-lysine-related radical with the same stereochemistry of hydrogen transfer from C2 of (S)-beta-lysine to the 5'-deoxyadenosyl radical as in the action of the clostridial enzyme. The resulting beta-lysyl radical has a conformation different from that at the active site of clostridial LAM. All evidence indicates that the opposite stereochemistry displayed by E. coli LAM is determined by the conformation of the lysine side chain in the active site. Stereochemical models for the actions of LAM from C. subterminale and E. coli are presented.

Identification of Structural and Catalytic Classes of Highly Conserved Amino Acid Residues in Lysine 2,3-Aminomutase

Lysine 2,3-aminomutase (LAM) from Clostridium subterminale SB4 catalyzes the interconversion of (S)-lysine and (S)-beta-lysine by a radical mechanism involving coenzymatic actions of S-adenosylmethionine (SAM), a [4Fe-4S] cluster, and pyridoxal 5'-phosphate (PLP). The enzyme contains a number of conserved acidic residues and a cysteine- and arginine-rich motif, which binds iron and sulfide in the [4Fe-4S] cluster. The results of activity and iron, sulfide, and PLP analysis of variants resulting from site-specific mutations of the conserved acidic residues and the arginine residues in the iron-sulfide binding motif indicate two classes of conserved residues of each type. Mutation of the conserved residues Arg134, Asp293, and Asp330 abolishes all enzymatic activity. On the basis of the X-ray crystal structure, these residues bind the epsilon-aminium and alpha-carboxylate groups of (S)-lysine. However, among these residues, only Asp293 appears to be important for stabilizing the [4Fe-4S] cluster. Members of a second group of conserved residues appear to stabilize the structure of LAM. Mutations of arginine 130, 135, and 136 and acidic residues Glu86, Asp165, Glu236, and Asp172 dramatically decrease iron and sulfide contents in the purified variants. Mutation of Asp96 significantly decreases iron and sulfide content. Arg130 or Asp172 variants display no detectable activity, whereas variants mutated at the other positions display low to very low activities. Structural roles are assigned to this latter class of conserved amino acids. In particular, a network of hydrogen bonded interactions of Arg130, Glu86, Arg135, and the main chain carbonyl groups of Cys132 and Leu55 appears to stabilize the [4Fe-4S] cluster.

How an Enzyme Tames Reactive Intermediates: Positioning of the Active-Site Components of Lysine 2,3-Aminomutase during Enzymatic Turnover As Determined by ENDOR Spectroscopy

Lysine 2,3-aminomutase (LAM) utilizes a [4Fe-4S] cluster, S-adenosyl-L-methionine (SAM), and pyridoxal 5'-phosphate (PLP) to isomerize L-alpha-lysine to L-beta-lysine. LAM is a member of the radical-SAM enzyme superfamily in which a [4Fe-4S]+ cluster reductively cleaves SAM to produce the 5'-deoxyadenosyl radical, which abstracts an H-atom from substrate to form 5'-deoxyadenosine (5'-Ado) and the alpha-Lys* radical (state 3 (Lys*)). This radical isomerizes to the beta-Lys* radical (state 4(Lys*)), which then abstracts an H-atom from 5'-Ado to form beta-lysine and the 5'-deoxyadenosyl radical; the latter then regenerates SAM. We use 13C, 1,2H, 31P, and 14N ENDOR to characterize the active site of LAM in intermediate states that contain the isomeric substrate radicals or analogues. With L-alpha-lysine as substrate, we monitor the state with beta-Lys*. In parallel, we use two substrate analogues that generate stable analogues of the alpha-Lys* radical: trans-4,5-dehydro-L-lysine (DHLys) and 4-thia-L-lysine (SLys). This first glimpse of the motions of active-site components during catalytic turnover suggests a possible major movement of PLP during catalysis. However, the principal focus of this work is on the relative positions of the carbons involved in H-atom transfer. We conclude that the active site facilitates hydrogen atom transfer by enforcing van der Waals contact between radicals and their reacting partners. This constraint enables the enzyme to minimize and even eliminate side reactions of highly reactive species such as the 5'-deoxyadensosyl radical.

Cofactor dependence of reduction potentials for [4Fe-4S]2+/1+ in lysine 2,3-aminomutase.

Lysine 2,3-aminomutase (LAM) catalyzes the interconversion of l-lysine and l-beta-lysine by a free radical mechanism. The 5'-deoxyadenosyl radical derived from the reductive cleavage of S-adenosyl-l-methionine (SAM) initiates substrate-radical formation. The [4Fe-4S](1+) cluster in LAM is the one-electron source in the reductive cleavage of SAM, which is directly ligated to the unique iron site in the cluster. We here report the midpoint reduction potentials of the [4Fe-4S](2+/1+) couple in the presence of SAM, S-adenosyl-l-homocysteine (SAH), or 5'-{N-[(3S)-3-aminocarboxypropyl]-N-methylamino}-5'-deoxyadenosine (azaSAM) as measured by spectroelectrochemistry. The reduction potentials are -430 +/- 2 mV in the presence of SAM, -460 +/- 3 mV in the presence of SAH, and -497 +/- 10 mV in the presence of azaSAM. In the absence of SAM or an analogue and the presence of dithiothreitol, dihydrolipoate, or cysteine as ligands to the unique iron, the midpoint potentials are -479 +/- 5, -516 +/- 5, and -484 +/- 3 mV, respectively. LAM is a member of the radical SAM superfamily of enzymes, in which the CxxxCxxC motif donates three thiolate ligands to iron in the [4Fe-4S] cluster and SAM donates the alpha-amino and alpha-carboxylate groups of the methionyl moiety as ligands to the fourth iron. The results show the reduction potentials in the midrange for ferredoxin-like [4Fe-4S] clusters. They show that SAM elevates the reduction potential by 86 mV relative to that of dihydrolipoate as the cluster ligand. This difference accounts for the SAM-dependent reduction of the [4Fe-4S](2+) cluster by dithionite reported earlier. Analogues of SAM have a weakened capacity to raise the potential. We conclude that the midpoint reduction potential of the cluster ligated to SAM is 1.2 V less negative than the half-wave potential for the one-electron reductive cleavage of simple alkylsulfonium ions in aqueous solution. The energetic barrier in the reductive cleavage of SAM may be overcome through the use of binding energy.

The x-ray crystal structure of lysine-2,3-aminomutase from Clostridium subterminale.

The x-ray crystal structure of the pyridoxal-5'-phosphate (PLP), S-adenosyl-L-methionine (SAM), and [4Fe-4S]-dependent lysine-2,3-aminomutase (LAM) of Clostridium subterminale has been solved to 2.1-A resolution by single-wavelength anomalous dispersion methods on a L-selenomethionine-substituted complex of LAM with [4Fe-4S]2+, PLP, SAM, and L-alpha-lysine, a very close analog of the active Michaelis complex. The unit cell contains a dimer of hydrogen-bonded, domain-swapped dimers, the subunits of which adopt a fold that contains all three cofactors in a central channel defined by six beta/alpha structural units. Zinc coordination links the domain-swapped dimers. In each subunit, the solvent face of the channel is occluded by an N-terminal helical domain, with the opposite end of the channel packed against the domain-swapped subunit. Hydrogen-bonded ionic contacts hold the external aldimine of PLP and L-alpha-lysine in position for abstraction of the 3-pro-R hydrogen of lysine by C5' of SAM. The structure of the SAM/[4Fe-4S] complex confirms and extends conclusions from spectroscopic studies of LAM and shows selenium in Se-adenosyl-L-selenomethionine poised to ligate the unique iron in the [4Fe-4S] cluster upon electron transfer and radical formation. The chain fold in the central domain is in part analogous to other radical-SAM enzymes.

Coordination and Mechanism of Reversible Cleavage of S-Adenosylmethionine by the [4Fe-4S] Center in Lysine 2,3-Aminomutase

Lysine 2,3-aminomutase (LAM) catalyzes the interconversion of l-lysine and l-beta-lysine, by a radical mechanism initiated by the reversible, reductive homolytic scission of the C5'-S bond in S-adenosylmethionine (SAM) to form methionine and the 5'-deoxyadenosyl radical at the active site. LAM is a member of a superfamily of enzymes in which a [4Fe-4S]+ cluster with a unique, noncysteinyl coordinated Fe provides the electron required in the cleavage of SAM. Little is known of the mechanism by which the electron is inserted into SAM, and it is not known whether all enzymes of the family employ the same mechanism. Selenium X-ray absorption spectroscopy (XAS) in the reaction of Se-adenosyl-l-selenomethionine (SeSAM) in place of SAM shows that electron transfer occurs by an inner sphere mechanism culminating in direct ligation of selenomethionine to iron upon cleavage of SeSAM. Here, we report an electron nuclear double resonance (ENDOR) spectroscopic investigation of LAM to which has been bound 14N, 17O, 2H, or 13C labeled SAM. It is found that LAM exhibits the same motif for SAM binding to the [4Fe-4S]+,2+ clusters as does pyruvate formate lyase: chelation by the unique iron of the amino and carboxylato groups of SAM; close proximity of the methionine methyl group to the cluster. However, there appear to be significant, and possibly mechanistically important, differences in the details of the binding geometry of SAM. On the basis of the correlation of the ENDOR and XAS spectroscopic results, we postulate a mechanism by which LAM cleaves SAM to generate an intermediate where N, O, and S of the methionine product are bound to the octahedrally coordinated unique Fe of the [4Fe-4S] cluster.

Characterization of an allylic analogue of the 5'-deoxyadenosyl radical: an intermediate in the reaction of lysine 2,3-aminomutase.

An allylic analogue of the 5'-deoxyadenosyl radical has been characterized at the active site of lysine 2,3-aminomutase (LAM) by electron paramagnetic resonance (EPR) spectroscopy. The anhydroadenosyl radical, 5'-deoxy-3',4'-anhydroadenosine-5'-yl, is a surrogate of the less stable 5'-deoxyadenosyl radical, which has never been observed but has been postulated to be a radical intermediate in the catalytic cycles of a number of enzymes. An earlier communication [Magnusson, O.Th., Reed, G. H., and Frey, P. A. (1999) J. Am. Chem. Soc. 121, 9764-9765] included the initial spectroscopic identification at 77 K of the radical, which is formed upon replacement of S-adenosylmethionine by S-3',4'-anhydroadenosylmethionine as a coenzyme for LAM. The electron paramagnetic resonance spectrum of the radical changes dramatically between 77 and 4.5 K. This unusual temperature dependence is attributed to a spin-spin interaction between the radical and thermally populated, higher spin states of the [4Fe-4S]+2 center, which is diamagnetic at 4.5 K. The EPR spectra of the radical at 4.5 K have been analyzed using isotopic substitutions and simulations. Analysis of the nuclear hyperfine splitting shows that the unpaired spin is distributed equally between C5'- and C3'- as expected for an allylic radical. Hyperfine splitting from the beta-proton at C-2'(H) shows that the dihedral angle to the p(z)-orbital at C-3' is approximately 37 degrees. This conformation is in good agreement with a structural model of the radical. The rate of formation of the allylic radical shows that it is kinetically competent as an intermediate. Measurements of 2H kinetic isotope effects indicate that with lysine as the substrate, the rate-limiting steps follow initial reductive cleavage of the coenzyme analogue.

Radical mechanisms of enzymatic catalysis.

Two classes of enzymatic mechanisms that proceed by free radical chemistry initiated by the 5'-deoxyadenosyl radical are discussed. In the first class, the mechanism of the interconversion of L-lysine and L-beta-lysine catalyzed by lysine 2,3-aminomutase (LAM) involves four radicals, three of which have been spectroscopically characterized. The reversible formation of the 5'-deoxyadenosyl radical takes place by the chemical cleavage of S-adenosylmethionine (SAM) reacting with the [4Fe-4S]+ center in LAM. In other reactions of SAM with iron-sulfur proteins, SAM is irreversibly consumed to generate the 5'-deoxyadenosyl radical, which activates an enzyme by abstracting a hydrogen atom from an enzymatic glycyl residue to form a glycyl radical. The glycyl radical enzymes include pyruvate formate-lyase, anaerobic ribonucleotide reductase from Escherichia coli, and benzylsuccinate synthase. Biotin synthase and lipoate synthase are SAM-dependent [4Fe-4S] proteins that catalyze the insertion of sulfur into unactivated C-H bonds, which are cleaved by the 5'-deoxyadenosyl radical from SAM. In the second class of enzymatic mechanisms using free radicals, adenosylcobalamin-dependent reactions, the 5'-deoxyadenosyl radical arises from homolytic cleavage of the cobalt-carbon bond, and it initiates radical reactions by abstracting hydrogen atoms from substrates. Three examples are described of suicide inactivation through the formation of exceptionally stable free radicals at enzymatic active sites.

The role of radicals in enzymatic processes

Research on the mechanism of action of coenzyme B12, adenosylcobalamin, as a graduate student introduced the author to the field of organic free radicals in enzymology. Twenty years later, related work on S-adenosylmethionine (SAM) as a "poor man's coenzyme B12" was initiated in a detailed analysis of the mechanism of action of lysine 2,3-aminomutase (LAM). The interconversion of L-lysine and L-beta-lysine is catalyzed by LAM, which requires SAM, pyridoxal-5'-phosphate (PLP), and a [4Fe-4S] cluster as coenzymes. The mechanism of this reaction has been delineated as a radical isomerization, in which radical formation is initiated by the [4Fe-4S]-dependent cleavage of the SAM into methionine and the 5'-deoxyadenosyl radical. The mechanism of this process is discussed, together with the role of this radical in hydrogen abstraction from lysine to initiate the substrate radical isomerization. The chemistry underlying the functions of SAM, PLP, and [4Fe-4S] in the action of LAM is novel in all respects, except for the formation of a lysine-PLP aldimine at the active site. Of the four free radicals in the mechanism, three have been characterized by EPR spectroscopy. In the suicide inactivation of adenosylcobalamin-dependent dioldehydrase (DDH) by glycolaldehyde, the formation of cob(II)alamin and 5'-deoxyadenosine is accompanied by the conversion of glycolaldehyde to cis-ethanesemidione radical at the active site. The cis-ethanesemidione radical has been characterized by EPR spectroscopy. Its exceptional stability at the active site is the basis for the inactivation of DDH by glycolaldehyde.

Identification of lysine 346 as a functionally important residue for pyridoxal 5'-phosphate binding and catalysis in lysine 2, 3-aminomutase from Bacillus subtilis.

Lysine 2,3-aminomutase (LAM) catalyzes the interconversion of L-lysine and L-beta-lysine. The enzyme contains pyridoxal 5'-phosphate (PLP) and a [4Fe-4S] center and requires S-adenosylmethionine (SAM) for activity. The hydrogen transfer is mediated by the 5'-deoxyadenosyl radical generated in a reaction of the iron-sulfur cluster with SAM. PLP facilitates the radical rearrangement by forming a lysine-PLP aldimine, in which the imine group participates in the isomerization mechanism. We here report the identification of lysine 346 as important for PLP binding and catalysis. Reduction of LAM with NaBH(4) rapidly inactivated the enzyme with concomitant UV/visible spectrum changes characteristic of reduction of an aldimine formed between PLP and lysine. Following reduction with NaBH(4) and proteolysis with trypsin, a single phosphopyridoxyl peptide of 36 amino acid residues was identified by reverse-phase liquid chromatography/mass spectrometry (LC/MS). The purified phosphopyridoxyl peptide exhibited an absorption band at 325 nm, and its identity was further confirmed by tandem mass spectrometry (MS/MS) sequencing. The bound PLP is linked to lysine 346 in a PGGGGK (PLP) structure. The sequence of this binding motif is conserved in LAMs from Bacillus and Clostridium and other homologous proteins but is distinct from the PLP-binding motifs found in other PLP enzymes. The function of lysine 346 was further studied by site-directed mutagenesis. The purified K346Q mutant was inactive, and its content of PLP was only approximately 15% of that of the wild-type enzyme. The data indicate that the formation of the aldimine linkage between lysine 346 and PLP is important for LAM catalysis. Sequences similar to the PLP-binding motifs in other enzymes were also present in LAM. However, lysine residues within these motifs neither are the PLP-binding sites in LAM nor are directly involved in LAM catalysis. This study represents the first comprehensive investigation of PLP binding in a SAM-dependent iron-sulfur enzyme.