A nucleosidase required for in vivo function of the S-adenosyl-L-methionine radical enzyme, biotin synthase

Biotin synthase is an S-adenosyl-L-methionine (SAM) radical enzyme that inserts sulfur into dethiobiotin to produce biotin. The reaction proceeds through 5'-deoxyadenosyl radical intermediates that become reduced during the sulfur insertion step to give another product of the reaction, 5'-deoxyadenosine. We report that Escherichia coli strains lacking the 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase encoded by the pfs gene are deficient in biotin synthase activity due to accumulation of 5'-deoxyadenosine, a new substrate of the pfs-encoded nucleosidase. Physiological experiments indicate that lipoic acid synthase, another SAM radical enzyme, is also inhibited by 5'-deoxyadenosine accumulation.     

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.     

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.     

Expanding Radical SAM Chemistry by Using Radical Addition Reactions and SAM Analogues

Radical S‐adenosyl‐l ‐methionine (SAM) enzymes utilize a [4Fe‐4S] cluster to bind SAM and reductively cleave its carbon–sulfur bond to produce a highly reactive 5′‐deoxyadenosyl (dAdo) radical. In almost all cases, the dAdo radical abstracts a hydrogen atom from the substrates or from enzymes, thereby initiating a highly diverse array of reactions. Herein, we report a change of the dAdo radical‐based chemistry from hydrogen abstraction to radical addition in the reaction of the radical SAM enzyme NosL. This change was achieved by using a substrate analogue containing an olefin moiety. We also showed that two SAM analogues containing different nucleoside functionalities initiate the radical‐based reactions with high efficiencies. The radical adduct with the olefin produced in the reaction was found to undergo two divergent reactions, and the mechanistic insights into this process were investigated in detail. Our study demonstrates a promising strategy in expanding radical SAM chemistry, providing an effective way to access nucleoside‐containing compounds by using radical SAM‐dependent reactions.     

Dihydroceramide delta(4) desaturase initiates substrate oxidation at C-4.

The intermolecular primary deuterium isotope effects on the individual C-H bond cleavage steps involved in dihydroceramide Delta(4) desaturation have been determined for the first time by incubating rat liver microsomes with 1:1 mixtures of nonlabeled substrate and the appropriate regiospecifically dideuterated analogue. Analysis of the enzymatic products via gas chromatography coupled to mass spectrometry showed that the introduction of the (E) double bond between C-4 and C-5 occurs in two discrete steps: cleavage of the C4-H bond was found to be very sensitive to isotopic substitution (k(H)/k(D) = 8.0 +/- 0.8), while a negligible isotope effect (k(H)/k(D) = 1.02 +/- 0.07) was observed for the C5-H bond-breaking step. According to a mechanistic model that we have previously proposed, these results suggest that initial oxidation for this desaturation reaction occurs at C-4. This finding correlates nicely with the observation that 4-hydroxylated products are produced from a similar substrate by a closely related oxidative enzyme in yeast.     

Substrate‐Tuned Catalysis of the Radical S‐Adenosyl‐L‐Methionine Enzyme NosL Involved in Nosiheptide Biosynthesis

NosL is a radical S‐adenosyl‐L ‐methionine (SAM) enzyme that converts L ‐Trp to 3‐methyl‐2‐indolic acid, a key intermediate in the biosynthesis of a thiopeptide antibiotic nosiheptide. In this work we investigated NosL catalysis by using a series of Trp analogues as the molecular probes. Using a benzofuran substrate 2‐amino‐3‐(benzofuran‐3‐yl)propanoic acid (ABPA), we clearly demonstrated that the 5′‐deoxyadenosyl (dAdo) radical‐mediated hydrogen abstraction in NosL catalysis is not from the indole nitrogen but likely from the amino group of L ‐Trp. Unexpectedly, the major product of ABPA is a decarboxylated compound, indicating that NosL was transformed to a novel decarboxylase by an unnatural substrate. Furthermore, we showed that, for the first time to our knowledge, the dAdo radical‐mediated hydrogen abstraction can occur from an alcohol hydroxy group. Our study demonstrates the intriguing promiscuity of NosL catalysis and highlights the potential of engineering radical SAM enzymes for novel activities.     

Revisiting the Mechanism of the Anaerobic Coproporphyrinogen III Oxidase HemN

HemN is a radical S‐adenosyl‐l ‐methionine (SAM) enzyme that catalyzes the oxidative decarboxylation of coproporphyrinogen III to produce protoporphyrinogen IX, an intermediate in heme biosynthesis. HemN binds two SAM molecules in the active site, but how these two SAMs are utilized for the sequential decarboxylation of the two propionate groups of coproporphyrinogen III remains largely elusive. Provided here is evidence showing that in HemN catalysis a SAM serves as a hydrogen relay which mediates a radical‐based hydrogen transfer from the propionate to the 5′‐deoxyadenosyl (dAdo) radical generated from another SAM in the active site. Also observed was an unexpected shunt product resulting from trapping of the SAM‐based methylene radical by the vinyl moiety of the mono‐decarboxylated intermediate, harderoporphyrinogen. These results suggest a major revision of the HemN mechanism and reveal a new paradigm of the radical‐mediated hydrogen transfer in radical SAM enzymology.     

Mechanistic Insights into the Radical S‐adenosyl‐l‐methionine Enzyme NosL From a Substrate Analogue and the Shunt Products

The radical S‐adenosyl‐l ‐methionine (SAM) enzyme NosL catalyzes the transformation of l ‐tryptophan into 3‐methyl‐2‐indolic acid (MIA), which is a key intermediate in the biosynthesis of a clinically interesting antibiotic nosiheptide. NosL catalysis was investigated by using the substrate analogue 2‐methyl‐3‐(indol‐3‐yl)propanoic acid (MIPA), which can be converted into MIA by NosL. Biochemical assays with different MIPA isotopomers in D₂O and H₂O unambiguously indicated that the 5′‐deoxyadenosyl (dAdo)‐radical‐mediated hydrogen abstraction is from the amino group of l ‐tryptophan and not a protein residue. Surprisingly, the dAdo‐radical‐mediated hydrogen abstraction occurs at two different sites of MIPA, thereby partitioning the substrate into different reaction pathways. Together with identification of an α,β‐unsaturated ketone shunt product, our study provides valuable mechanistic insight into NosL catalysis and highlights the remarkable catalytic flexibility of radical SAM enzymes.     

Probing the reaction mechanism of spore photoproduct lyase (SPL) via diastereoselectively labeled dinucleotide SP TpT substrates

5-Thyminyl-5,6-dihydrothymine (commonly called spore photoproduct or SP) is the exclusive DNA photodamage product in bacterial endospores. It is generated in the bacterial sporulation phase and repaired by a radical SAM enzyme, spore photoproduct lyase (SPL), at the early germination phase. SPL utilizes a special [4Fe-4S] cluster to reductively cleave S-adenosylmethionine (SAM) to generate a reactive 5'-dA radical. The 5'-dA radical is proposed to abstract one of the two H-atoms at the C6 carbon of SP to initiate the repair process. Via organic synthesis and DNA photochemistry, we selectively labeled the 6-H(proS) or 6-H(proR) position with a deuterium in a dinucleotide SP TpT substrate. Monitoring the deuterium migration in enzyme catalysis (employing Bacillus subtilis SPL) revealed that it is the 6-H(proR) atom of SP that is abstracted by the 5'-dA radical. Surprisingly, the abstracted deuterium was not returned to the resulting TpT after enzymatic catalysis; an H-atom from the aqueous buffer was incorporated into TpT instead. This result questions the currently hypothesized SPL mechanism which excludes the involvement of protein residue(s) in SPL reaction, suggesting that some protein residue(s), which is capable of exchanging a proton with the aqueous buffer, is involved in the enzyme catalysis. Moreover, evidence has been obtained for a possible SAM regeneration after each catalytic cycle; however, such a regeneration process is more complex than currently thought, with one or even more protein residues involved as well. These observations have enabled us to propose a modified reaction mechanism for this intriguing DNA repair enzyme.     

Aliphatic peptidyl hydroperoxides as a source of secondary oxidation in hydroxyl radical protein footprinting

Hydroxyl radical footprinting is a technique for studying protein structure and binding that entails oxidizing a protein system of interest with diffusing hydroxyl radicals, and then measuring the amount of oxidation of each amino acid. One important issue in hydroxyl radical footprinting is limiting amino acid oxidation by secondary oxidants to prevent uncontrolled oxidation, which can cause amino acids to appear more solvent accessible than they really are. Previous work suggested that hydrogen peroxide was the major secondary oxidant of concern in hydroxyl radical footprinting experiments; however, even after elimination of all hydrogen peroxide, some secondary oxidation was still detected. Evidence is presented for the formation of peptidyl hydroperoxides as the most abundant product upon oxidation of aliphatic amino acids. Both reverse phase liquid chromatography and catalase treatment were shown to be ineffective at eliminating peptidyl hydroperoxides. The ability of these peptidyl hydroperoxides to directly oxidize methionine is demonstrated, suggesting the value of methionine amide as an in situ protectant. Hydroxyl radical footprinting protocols require the use of an organic sulfide or similar peroxide scavenger in addition to removal of hydrogen peroxide to successfully eradicate all secondary oxidizing species and prevent uncontrolled oxidation of sulfur-containing residues.     

Computational insights into the mechanism of radical generation in B12-dependent methylmalonyl-CoA mutase

ONIOM calculations have provided novel insights into the mechanism of homolytic Co-C5' bond cleavage in the 5'-deoxyadenosylcobalamin cofactor catalyzed by methylmalonyl-CoA mutase. We have shown that it is a stepwise process in which conformational changes in the 5'-deoxyadenosine moiety precede the actual homolysis step. In the transition state structure for homolysis, the Co-C5' bond elongates by approximately 0.5 Angstroms from the value found in the substrate-bound reactant complex. The overall barrier to homolysis is approximately 10 kcal/mol, and the radical products are approximately 2.5 kcal/mol less stable than the initial ternary complex of enzyme, substrate, and cofactor. The movement of the deoxyadenosine moiety during the homolysis step positions the resulting 5'-deoxyadenosyl radical for the subsequent hydrogen atom transfer from the substrate, methylmalonyl-CoA.     

Characterization of Radical SAM Adenosylhopane Synthase,HpnH, which Catalyzes the 5′‐Deoxyadenosyl Radical Addition to Diploptene in the Biosynthesis of C35 Bacteriohopanepolyols

Adenosylhopane is a crucial intermediate in the biosynthesis of bacteriohopanepolyols, which are widespread prokaryotic membrane lipids. Herein, it is demonstrated that reconstituted HpnH, a putative radical S‐adenosyl‐l ‐methionine (SAM) enzyme, commonly encoded in the hopanoid biosynthetic gene cluster, converts diploptene into adenosylhopane in the presence of SAM, flavodoxin, flavodoxin reductase, and NADPH. NMR spectra of the enzymatic reaction product were identical to those of synthetic (22R)‐adenosylhopane, indicating that HpnH catalyzes stereoselective C?C formation between C29 of diploptene and C5′ of 5′‐deoxyadenosine. Further, the HpnH reaction in D₂O‐containing buffer revealed that a D atom was incorporated at the C22 position of adenosylhopane. Based on these results, we propose a radical addition reaction mechanism catalyzed by HpnH for the formation of the C₃₅ bacteriohopane skeleton.     

Homology Modeling and Molecular Docking Studies of (S)‐Scoulerine 9‐O‐Methyltransferase from Coptis chinensis

(S)‐Scoulerine 9‐O‐methyltransferase (SMT), belonging to the S‐adenosyl‐L‐methionine (SAM)‐dependent O‐methyltransferase family, is an essential enzyme in the berberine biosynthetic pathways. In order to study the interactions of SMT with its substrate and further to understand the catalytic mechanism and substrate specificity, a three dimensional model of SMT from Coptis chinensis was constructed by homology modeling using the crystal structure of caffeic acid/5‐hydroxyferulic acid 3/5‐O‐methyltransferase (COMT) as a template. The three dimensional structure of SMT, which was mainly composed of α‐helices and some β‐sheets, was similar to that of COMT. In contrast with COMT, the non‐conserved residues in the substrate binding pocket of SMT might be responsible for their differences in the substrate specificity. Val119 and Asp254 in SMT were the key residues for orienting substrate for methylation as both residues had H‐bonds with (S)‐scoulerine. The methylation of (S)‐scoulerine involved deprotonation of the 9‐hydroxyl group by His253 and Asp254 in SMT followed by a nucleophilic attack on the SAM‐methyl resulting in the product, (S)‐tetrahydrocolumbamine.     

Hydrogen Transfer in SAM‐Mediated Enzymatic Radical Reactions

S‐Adenosylmethionine (SAM) plays an essential role in a variety of enzyme‐mediated radical reactions. One‐electron reduction of SAM is currently believed to generate the C5′‐desoxyadenosyl radical, which subsequently abstracts a hydrogen atom from the actual substrate in a catalytic or a non‐catalytic fashion. Using a combination of theoretical and experimental bond dissociation energy (BDE) data, the energetics of these radical processes have now been quantified. SAM‐derived radicals are found to react with their respective substrates in an exothermic fashion in enzymes using SAM in a stoichiometric (non‐catalytic) way. In contrast, the catalytic use of SAM appears to be linked to a sequence of moderately endothermic and exothermic reaction steps. The use of SAM in spore photoproduct lyase (SPL) appears to fit neither of these general categories and appears to constitute the first example of a SAM‐initiated radical reaction propagated independently of the cofactor.     

C3′‐Deoxygenation of Paromamine Catalyzed by a Radical S‐Adenosylmethionine Enzyme: Characterization of the Enzyme AprD4 and Its Reductase Partner AprD3

C3′‐deoxygenation of aminoglycosides results in their decreased susceptibility to phosphorylation thereby increasing their efficacy as antibiotics. However, the biosynthetic mechanism of C3′‐deoxygenation is unknown. To address this issue, aprD4 and aprD3 genes from the apramycin gene cluster in Streptomyces tenebrarius were expressed in E. coli and the resulting gene products were characterized in vitro. AprD4 is shown to be a radical S‐adenosylmethionine (SAM) enzyme, catalyzing homolysis of SAM to 5′‐deoxyadenosine (5′‐dAdo) in the presence of paromamine. [4′‐²H]‐Paromamine was prepared and used to show that its C4′‐H is transferred to 5′‐dAdo by AprD4, during which the substrate is dehydrated to a product consistent with 4′‐oxolividamine. In contrast, paromamine is reduced to a deoxy product when incubated with AprD4/AprD3/NADPH. These results show that AprD4 is the first radical SAM diol‐dehydratase and, along with AprD3, is responsible for 3′‐deoxygenation in aminoglycoside biosynthesis.     

Conversion of methionine into homocysteic acid in heavily oxidized proteomics samples

Analysis of protein oxidation is necessary in numerous areas of biochemistry, including hydroxyl radical surface mapping, oxidative stress assays, and pharmaceutical stability testing. Mass spectrometry is one of the tools most often used to identify protein oxidation products, and previous studies have attempted to identify and characterize all of the major oxidation products detected by mass spectrometry for each amino acid residue. In this note, we present evidence that in heavily oxidized protein samples, such as those produced by hydroxyl radical surface mapping, a major oxidation product of methionine is homocysteic acid. The formation of homocysteic acid from methionine was previously unrecognized in other mass spectrometric analyses, and has important implications for the analysis of oxidized samples, as well as potential implications as to the functional consequences of methionine oxidation. Copyright © 2010 John Wiley & Sons, Ltd.     

Interaction of the substrate radical and the 5'-deoxyadenosine-5'-methyl group in vitamin B(12) coenzyme-dependent ethanolamine deaminase

The distance and relative orientation of the C5' methyl group of 5'-deoxyadenosine and the substrate radical in vitamin B(12) coenzyme-dependent ethanolamine deaminase from Salmonella typhimurium have been characterized by using X-band two-pulse electron spin-echo envelope modulation (ESEEM) spectroscopy in the disordered solid state. The (S)-2-aminopropanol-generated substrate radical catalytic intermediate was prepared by cryotrapping steady-state mixtures of enzyme in which catalytically exchangeable hydrogen sites in the active site had been labeled by previous turnover on (2)H(4)-ethanolamine. Simulation of the time- and frequency-domain ESEEM requires two types of coupled (2)H. The strongly coupled (2)H has an effective dipole distance (r(eff)) of 2.2 A, and isotropic coupling constant (A(iso)) of -0.35 MHz. The weakly coupled (2)H has r(eff) = 3.8 A and A(iso) = 0 MHz. The best (2)H ESEEM time- and frequency-domain simulations are achieved with a model in which the hyperfine couplings arise from one strongly coupled hydrogen site and two equivalent weakly coupled hydrogen sites located on the C5' methyl group of 5'-deoxyadenosine. This model indicates that the unpaired electron on C1 of the substrate radical and C5' are separated by 3.2 A and are thus at closest contact. The close proximity of C1 and C5' indicates that C5' of the 5'-deoxyadenosyl moiety directly mediates radical migration between cobalt in cobalamin and the substrate/product site over a distance of 5-7 A in the active site of ethanolamine deaminase.     

Local stability of Rhodobacter capsulatus cytochrome c 2 probed by solution phase hydrogen/deuterium exchange and mass spectrometry

The hydrogen/deuterium exchange kinetics of Rhodobacter capsulatus cytochrome c2 have been determined using mass spectrometry. As expected, the relative domain stability was generally similar to that of the cytochrome c2 structural homolog, horse heart cytochrome c, but we were able to find evidence to support the presence of a second, small beta-sheet not found in the horse cytochrome, which stabilizes a structural region dominated by Omega loops. Importantly, we find that the so-called hinge region, comprised of 15 amino acids, which include the methionine sixth heme ligand (M96), is destabilized on oxidation, and this destabilization is propagated to a portion of the second Omega loop, most likely through perturbation of two hydrogen bonds that couple these two domains in the three dimensional structure. The mutation of a lysine at position 93 to proline amplifies the destabilization observed on oxidation of the wild-type cytochrome c2 and results in further destabilization observed in regions 52-60, 75-82, and 83-97. This suggests that hydrogen bond interactions involving two bound waters, the T94 hydroxyl, the front heme propionate and the Y75 hydroxyl, are significantly compromised upon mutation. In summary, these observations are consistent with the approximately 20-fold increase in the movement of the hinge away from the heme face in the oxidized cytochrome c2 as determined by ligand binding kinetics. Thus, H/D exchange kinetics can be used to identify relatively subtle structural features and at least in some cases facilitate the understanding of the structural basis of the dynamic properties of proteins.     

Iron-sulfur proteins as initiators of radical chemistry

Iron-sulfur proteins are very versatile biological entities for which many new functions are continuously being unravelled. This review focus on their role in the initiation of radical chemistry, with special emphasis on radical-SAM enzymes, since several members of the family catalyse key steps in the biosynthetic pathways of cofactors such as biotin, lipoate, thiamine, heme and the molybdenum cofactor. It will also include other examples to show the chemical logic which is emerging from the presently available data on this family of enzymes. The common step in all the (quite different) reactions described here is the monoelectronic reductive cleavage of SAM by a reduced [4Fe-4S](1+) cluster, producing methionine and a highly oxidising deoxyadenosyl radical, which can initiate chemically difficult reactions. This set of enzymes, which represent a means to perform oxidation under reductive conditions, are often present in anaerobic organisms. Some other, non-SAM-dependent, radical reactions obeying the same chemical logic are also covered.     

Crystal Structure of Tryptophan Lyase (NosL): Evidence for Radical Formation at the Amino Group of Tryptophan

Streptomyces actuosus tryptophan lyase (NosL) is a radical SAM enzyme which catalyzes the synthesis of 3‐methyl‐2‐indolic acid, a precursor in the synthesis of the promising antibiotic nosiheptide. The reaction involves cleavage of the tryptophan Cα? Cβ bond and recombination of the amino‐acid‐derived ‐COOH fragment at the indole ring. Reported herein is the 1.8 Å resolution crystal structure of NosL complexed with its substrate. Unexpectedly, only one of the tryptophan amino hydrogen atoms is optimally placed for H abstraction by the SAM‐derived 5′‐deoxyadenosyl radical. This orientation, in turn, rules out the previously proposed delocalized indole radical as the species which undergoes Cα? Cβ bond cleavage. Instead, stereochemical considerations indicate that the reactive intermediate is a ^.NH tryptophanyl radical. A structure‐based amino acid sequence comparison of NosL with the tyrosine lyases ThiH and HydG strongly suggests that an equivalent ^.NH radical operates in the latter enzymes.