Molecular cloning and functional characterization of 2,3-oxidosqualene cyclases from Artemisia argyi.

Artemisia argyi is a traditional medicinal and edible plant, generating various triterpenoids with pharmacological activities, such as anti-virus, anti-cancer, and anti-oxidant. The 2,3-oxidosqualene cyclase family of A. argyi offers novel insights into the triterpenoid pathway, which might contribute to the medicinal value of its tissue extracts. Nevertheless, the biosynthesis of active triterpenoids in Artemisia argyi is still uncertain. In this study, four putative OSC (2,3-oxidosqualene cyclase) genes (AaOSC1-4) were first isolated and identified from A. argyi. Through the yeast heterologous expression system, three AaOSCs were characterized for the biosynthesis of diverse triterpenoids including cycloartenol, ß-amyrin, (3S,13R)-malabarica-14(27),17,21-trien-3ß-ol, and dammara-20,24-dien-3ß-ol. AaOSC1 was a multifunctional dammara-20,24-dien-3ß-ol synthase, which yielded 8 different triterpenoids, including tricyclic, and tetracyclic products. AaOSC2 and AaOSC3 were cycloartenol, and ß-amyrin synthases, respectively. As a result, these findings provide a deeper understanding of the biosynthesis pathway of triterpenes in A. argyi.

Structural basis of pri-let-7 recognition by human pseudouridine synthase TruB1.

Let-7 was one of the first microRNAs (miRNAs) to be discovered and its expression promotes differentiation during development and function as tumor suppressors in various cancers. The maturation process of let-7 miRNA is tightly regulated by multiple RNA-binding proteins. For example, LIN28 binds to the terminal loops of the precursors of let-7 family and block their processing into mature miRNAs. Trim25 promotes the uridylation-mediated degradation of pre-let-7 modified by LIN28/TUT4. Recently, human pseudouridine synthase TruB1 has been reported to facilitate let-7 maturation by directly binding to pri-let-7 and recruiting Drosha-DGCR8 microprocessor. Through biochemical assay and structural investigation, we show that human TruB1 binds specifically the terminal loop of pri-let-7a1 at nucleotides 31-41, which folds as a small stem-loop architecture. Although TruB1 recognizes the terminal loop of pri-let-7a1 in a way similar to how E. coli TruB interacts with tRNA, a conserved KRKK motif in human and other higher eukaryotes adds an extra binding interface and strengthens the recognition of TruB1 for pri-let-7a1 through electrostatic interactions. These findings reveal the structural basis of TruB1-pri-let-7 interaction which may assists the elucidation of precise role of TruB1 in biogenesis of let-7.

Genome-Wide Investigation of Oxidosqualene Cyclase Genes Deciphers the Genetic Basis of Triterpene Biosynthesis in Tea Plants.

Triterpenoids from Camellia species comprise a diverse class of bioactive compounds with great therapeutic potential. However, triterpene biosynthesis in tea plants (Camellia sinensis) remains elusive. Here, we identified eight putative 2,3-oxidosqualene cyclase (OSC) genes (CsOSC1-8) from the tea genome and characterized the functions of five through heterologous expression in yeast and tobacco and transient overexpression in tea plants. CsOSC1 was found to be a ß-amyrin synthase, whereas CsOSC4, 5, and 6 exhibited multifunctional α-amyrin synthase activity. Molecular docking and site-directed mutagenesis showed that the CsOSC6M259T/W260L double mutant yielded >40% lupeol, while the CsOSC1 W259L single mutant alone was sufficient for lupeol production. The V732F mutation in CsOSC5 altered product formation from friedelin to taraxasterol and ψ-taraxasterol. The L254 M mutation in the cycloartenol synthase CsOSC8 enhanced the catalytic activity. Our findings shed light on the molecular basis governing triterpene diversity in tea plants and offer potential avenues for OSC engineering.

Targeting PUS7 suppresses tRNA pseudouridylation and glioblastoma tumorigenesis.

Pseudouridine is the most frequent epitranscriptomic modification. However, its cellular functions remain largely unknown. Here, we show that pseudouridine synthase 7 (PUS7) is highly expressed in glioblastoma versus normal brain tissues, and high PUS7 expression levels are associated with worse survival in patients with glioblastoma. PUS7 expression and catalytic activity are required for glioblastoma stem cell (GSC) tumorigenesis. Mechanistically, we identify PUS7 targets in GSCs through small RNA pseudouridine sequencing and show that pseudouridylation of PUS7-regulated transfer RNA is critical for codon-specific translational control of key regulators of GSCs. Moreover, we identify chemical inhibitors for PUS7 and show that these compounds prevent PUS7-mediated pseudouridine modification, suppress tumorigenesis and extend the life span of tumor-bearing mice. Overall, we identify an epitranscriptomic regulatory mechanism in glioblastoma and provide preclinical evidence of a potential therapeutic strategy for glioblastoma.

An Unprecedented Ring-Contraction Mechanism in Cobalamin-Dependent Radical S-Adenosylmethionine Enzymes.

A unique member of the family of cobalamin (Cbl)-dependent radical S-adenosylmethionine (SAM) enzymes, OxsB, catalyzes the ring constriction of deoxyadenosine triphosphate (dATP) to the base oxetane aldehyde phosphate, a crucial precursor for oxetanocin A (OXT-A), which is an antitumor, antiviral, and antibacterial compound. This enzyme reveals a new catalytic function for this big family that is different from the common methylation. On the basis of density functional theory calculations, a mechanism has been proposed to mainly include that the generation of 5'-deoxyadenosine radical, a hydrogen transfer forming 2'-dATP radical, and a Cbl-catalyzed ring contraction of the deoxyribose in 2'-dATP radical. The ring contraction is a concerted rearrangement step accompanied by an electron transfer from the deoxyribose hydroxyl oxygen to CoIII without any ring-opening intermediate. CoIICbl has been ruled out as an active state. Other mechanistic characteristics are also revealed. This unprecedented non-methylation mechanism provides a new catalytic repertoire for the family of radical SAM enzymes, representing a new class of ring-contraction enzymes.

Molecular characteristics of plant UDP-arabinopyranose mutases.

l-arabinofuranose is a ubiquitous component of the cell wall and various natural products in plants, where it is synthesized from cytosolic UDP-arabinopyranose (UDP-Arap). The biosynthetic machinery long remained enigmatic in terms of responsible enzymes and subcellular localization. With the discovery of UDP-Arap mutase in plant cytosol, the demonstration of its role in cell-wall arabinose incorporation and the identification of UDP-arabinofuranose transporters in the Golgi membrane, it is clear that the cytosolic UDP-Arap mutases are the key enzymes converting UDP-Arap to UDP-arabinofuranose for cell wall and natural product biosynthesis. This has recently been confirmed by several genotype/phenotype studies. In contrast to the solid evidence pertaining to UDP-Arap mutase function in vivo, the molecular features, including enzymatic mechanism and oligomeric state, remain unknown. However, these enzymes belong to the small family of proteins originally identified as reversibly glycosylated polypeptides (RGPs), which has been studied for >20 years. Here, we review the UDP-Arap mutase and RGP literature together, to summarize and systemize reported molecular characteristics and relations to other proteins.

Human Acetyl-CoA Carboxylase 1 Is an Isomerase: Carboxyl Transfer Is Activated by Catalytic Effect of Isomerization.

Obesity and its related diseases such as cancer and diabetes are leading life-threatening issues in the modern world. Thus, new drugs toward obesity and obesity-caused diseases are highly desired. Human acetyl-CoA carboxylase 1 (hACC1) in charge of the rate-limiting step of the human fatty acid synthesis was recognized as an attractive target for rational drug design. The fundamental reaction mechanism and nature of the transition state of hACC1 remain unclear. In this study, combined quantum mechanics and molecular mechanics (QM/MM), molecular dynamics (MD), and free-energy simulations were performed to investigate the catalytic mechanism of the hACC1-catalyzed carboxyl-transfer reaction. Our computational results show a three-step mechanism for carboxyl transferase (CT)-catalyzed reaction, including isomerization of carboxybiotin, proton-transfer from acetyl-CoA to carboxybiotin, and carboxylation of acetyl-CoA enolate. Interestingly, isomerization of carboxybiotin is the rate-limiting step of the entire reaction pathway, indicating hACC1 has the catalytic effect of isomerization and thus might be an isomerase also. The activation free-energy barrier of carboxyl-transfer catalyzed by hACC1 was calculated to be 16.4 kcal/mol, in excellent agreement with the experimental result (16.7 kcal/mol). The obtained reaction mechanism together with the nature of the transition state provides helpful knowledge not only for future investigation of other ACCs but also for rational design of hACC1 inhibitors, such as TS analogue. The catalytic effect of hACC1 isomerization is discussed.

Identification of oxidosqualene cyclases from the medicinal legume tree Bauhinia forficata: a step toward discovering preponderant α-amyrin-producing activity.

Triterpenoids are widely distributed among plants of the legume family. However, most studies have focused on triterpenoids and their biosynthetic enzymes in model legumes. We evaluated the triterpenoid aglycones profile of the medicinal legume tree Bauhinia forficata by gas chromatography-mass spectrometry. Through transcriptome analyses, homology-based cloning, and heterologous expression, we discovered four oxidosqualene cyclases (OSCs) which are responsible for the diversity of triterpenols in B. forficata. We also investigated the effects of the unique motif TLCYCR on α-amyrin synthase activity. B. forficata highly accumulated α-amyrin. We discovered an OSC with a preponderant α-amyrin-producing activity, which accounted for at least 95% of the total triterpenols. We also discovered three other functional OSCs (BfOSC1, BfOSC2, and BfOSC4) that produce ß-amyrin, germanicol, and cycloartenol. Furthermore, by replacing the unique motif TLCYCR from BfOSC3 with the MWCYCR motif, we altered the function of BfOSC3 such that it no longer produced α-amyrin. Our results provide new insights into OSC cyclization, which is responsible for the diversity of triterpenoid metabolites in B. forficata, a non-model legume plant.

Magnetic field effects on coenzyme B12- and B6-dependent lysine 5,6-aminomutase: switching of the J-resonance through a kinetically competent radical-pair intermediate.

The environmental magnetic field is beneficial to migratory bird navigation through the radical-pair mechanism. One of the continuing challenges in understanding how magnetic fields may perturb biological processes is that only a very few field-sensitive examples have been explored despite the prevalence of radical pairs in enzymatic reactions. We show that the reaction of adenosylcobalamin- and pyridoxal-5'-phosphate-dependent lysine 5,6-aminomutase proceeds via radical-pair intermediates and is magnetic field dependent. The 5'-deoxyadenosyl radical from adenosylcobalamin abstracts a C5(H) from the substrate to yield a {cob(ii)alamin - substrate} radical pair wherein the large spin-spin interaction (2J = 8000 gauss) locks the radical pair in a triplet state, as evidenced by electron paramagnetic resonance spectroscopy. Application of an external magnetic field in the range of 6500 to 8500 gauss triggers intersystem crossing to the singlet {cob(ii)alamin - substrate} radical-pair state. Spin-conserved H back-transfer from deoxyadenosine to the substrate radical yields a singlet {cob(ii)alamin-5'-deoxyadenosyl} radical pair. Spin-selective recombination to adenosylcobalamin decreased the enzyme catalytic efficiency kcat/Km by 16% at 7600 gauss. As a mechanistic probe, observation of magnetic field effects successfully demonstrates the presence of a kinetically significant radical pair in this enzyme. The study of a pronounced high-field level-crossing characteristic through an immobilized radical pair with a constant exchange interaction deepens our understanding of how a magnetic field may interact with an enzyme.

Comparative analysis of plant isochorismate synthases reveals structural mechanisms underlying their distinct biochemical properties.

Isochorismate synthase (ICS) converts chorismate into isochorismate, a precursor of primary and secondary metabolites including salicylic acid (SA). SA plays important roles in responses to stress conditions in plants. Many studies have suggested that the function of plant ICSs is regulated at the transcriptional level. In Arabidopsis thaliana, the expression of AtICS1 is induced by stress conditions in parallel with SA synthesis, and AtICS1 is required for SA synthesis. In contrast, the expression of NtICS is not induced when SA synthesis is activated in tobacco, and it is unlikely to be involved in SA synthesis. Studies on the biochemical properties of plant ICSs are limited, compared with those on transcriptional regulation. We analyzed the biochemical properties of four plant ICSs: AtICS1, NtICS, NbICS from Nicotiana benthamiana, and OsICS from rice. Multiple sequence alignment analysis revealed that their primary structures were well conserved, and predicted key residues for ICS activity were almost completely conserved. However, AtICS1 showed much higher activity than the other ICSs when expressed in Escherichia coli and N. benthamiana leaves. Moreover, the levels of AtICS1 protein expression in N. benthamiana leaves were higher than the other ICSs. Construction and analysis of chimeras between AtICS1 and OsICS revealed that the putative chloroplast transit peptides (TPs) significantly affected the levels of protein accumulation in N. benthamiana leaves. Chimeric and point-mutation analyses revealed that Thr531, Ser537, and Ile550 of AtICS1 are essential for its high activity. These distinct biochemical properties of plant ICSs may suggest different roles in their respective plant species.

Design strategies of oxidosqualene cyclase inhibitors: Targeting the sterol biosynthetic pathway.

Targeting the sterol biosynthesis pathway has been explored for the development of new bioactive compounds. Among the enzymes of this pathway, oxidosqualene cyclase (OSC) which catalyzes lanosterol cyclization from 2,3-oxidosqualene has emerged as an attractive target. In this work, we reviewed the most promising OSC inhibitors from different organisms and their potential for the development of new antiparasitic, antifungal, hypocholesterolemic and anticancer drugs. Different strategies have been adopted for the discovery of new OSC inhibitors, such as structural modifications of the natural substrate or the reaction intermediates, the use of the enzyme's structural information to discover compounds with novel chemotypes, modifications of known inhibitors and the use of molecular modeling techniques such as docking and virtual screening to search for new inhibitors. This review brings new perspectives on structural insights of OSC from different organisms and reveals the broad structural diversity of OSC inhibitors which may help evidence lead compounds for further investigations with various therapeutic applications.

X-ray diffraction analysis and in vitro characterization of the UAM2 protein from Oryza sativa.

The role of seemingly non-enzymatic proteins in complexes interconverting UDP-arabinopyranose and UDP-arabinofuranose (UDP-arabinosemutases; UAMs) in the plant cytosol remains unknown. To shed light on their function, crystallographic and functional studies of the seemingly non-enzymatic UAM2 protein from Oryza sativa (OsUAM2) were undertaken. Here, X-ray diffraction data are reported, as well as analysis of the oligomeric state in the crystal and in solution. OsUAM2 crystallizes readily but forms highly radiation-sensitive crystals with limited diffraction power, requiring careful low-dose vector data acquisition. Using size-exclusion chromatography, it is shown that the protein is monomeric in solution. Finally, limited proteolysis was employed to demonstrate DTT-enhanced proteolytic digestion, indicating the existence of at least one intramolecular disulfide bridge or, alternatively, a requirement for a structural metal ion.

Analysis of plant UDP-arabinopyranose mutase (UAM): Role of divalent metals and structure prediction.

UDP-arabinopyranose mutase (UAM) is a plant enzyme which interconverts UDP-arabinopyranose (UDP-Arap; a six-membered sugar) to UDP-arabinofuranose (UDP-Araf; a five-membered sugar). Plant mutases belong to a small gene family called Reversibly Glycosylated Proteins (RGPs). So far, UAM has been identified in Oryza sativa (Rice), Arabidopsis thaliana and Hordeum vulgare (Barley). The enzyme requires divalent metal ions for catalytic activity. Here, the divalent metal ion dependency of UAMs from O. sativa (rice) and A. thaliana have been studied using HPLC-based kinetic assays. It was determined that UAM from these species had the highest relative activity in a range of 40-80µM Mn2+. Excess Mn2+ ion concentration decreased the enzyme activity. This trend was observed when other divalent metal ions were used to test activity. To gain a perspective of the role played by the metal ion in activity, an ab initio structural model was generated based on the UAM amino acid sequence and a potential metal binding region was identified. Based on our results, we propose that the probable role of the metal in UAM is stabilizing the diphosphate of the substrate, UDP-Arap.

Structural Basis for Substrate Specificity in Adenosylcobalamin-dependent Isobutyryl-CoA Mutase and Related Acyl-CoA Mutases.

Acyl-CoA mutases are a growing class of adenosylcobalamin-dependent radical enzymes that perform challenging carbon skeleton rearrangements in primary and secondary metabolism. Members of this class of enzymes must precisely control substrate positioning to prevent oxidative interception of radical intermediates during catalysis. Our understanding of substrate specificity and catalysis in acyl-CoA mutases, however, is incomplete. Here, we present crystal structures of IcmF, a natural fusion protein variant of isobutyryl-CoA mutase, in complex with the adenosylcobalamin cofactor and four different acyl-CoA substrates. These structures demonstrate how the active site is designed to accommodate the aliphatic acyl chains of each substrate. The structures suggest that a conformational change of the 5'-deoxyadenosyl group from C2'-endo to C3'-endo could contribute to initiation of catalysis. Furthermore, detailed bioinformatic analyses guided by our structural findings identify critical determinants of acyl-CoA mutase substrate specificity and predict new acyl-CoA mutase-catalyzed reactions. These results expand our understanding of the substrate specificity and the catalytic scope of acyl-CoA mutases and could benefit engineering efforts for biotechnological applications ranging from production of biofuels and commercial products to hydrocarbon remediation.

Engineered and Native Coenzyme B12-dependent Isovaleryl-CoA/Pivalyl-CoA Mutase.

Adenosylcobalamin-dependent isomerases catalyze carbon skeleton rearrangements using radical chemistry. We have recently demonstrated that an isobutyryl-CoA mutase variant, IcmF, a member of this enzyme family that catalyzes the interconversion of isobutyryl-CoA and n-butyryl-CoA also catalyzes the interconversion between isovaleryl-CoA and pivalyl-CoA, albeit with low efficiency and high susceptibility to inactivation. Given the biotechnological potential of the isovaleryl-CoA/pivalyl-CoA mutase (PCM) reaction, we initially attempted to engineer IcmF to be a more proficient PCM by targeting two active site residues predicted based on sequence alignments and crystal structures, to be key to substrate selectivity. Of the eight mutants tested, the F598A mutation was the most robust, resulting in an ∼17-fold increase in the catalytic efficiency of the PCM activity and a concomitant ∼240-fold decrease in the isobutyryl-CoA mutase activity compared with wild-type IcmF. Hence, mutation of a single residue in IcmF tuned substrate specificity yielding an ∼4000-fold increase in the specificity for an unnatural substrate. However, the F598A mutant was even more susceptible to inactivation than wild-type IcmF. To circumvent this limitation, we used bioinformatics analysis to identify an authentic PCM in genomic databases. Cloning and expression of the putative AdoCbl-dependent PCM with an α2ß2 heterotetrameric organization similar to that of isobutyryl-CoA mutase and a recently characterized archaeal methylmalonyl-CoA mutase, allowed demonstration of its robust PCM activity. To simplify kinetic analysis and handling, a variant PCM-F was generated in which the αß subunits were fused into a single polypeptide via a short 11-amino acid linker. The fusion protein, PCM-F, retained high PCM activity and like PCM, was resistant to inactivation. Neither PCM nor PCM-F displayed detectable isobutyryl-CoA mutase activity, demonstrating that PCM represents a novel 5'-deoxyadenosylcobalamin-dependent acyl-CoA mutase. The newly discovered PCM and the derivative PCM-F, have potential applications in bioremediation of pivalic acid found in sludge, in stereospecific synthesis of C5 carboxylic acids and alcohols, and in the production of potential commodity and specialty chemicals.

Structural basis of the stereospecificity of bacterial B12-dependent 2-hydroxyisobutyryl-CoA mutase.

Bacterial coenzyme B12-dependent 2-hydroxyisobutyryl-CoA mutase (HCM) is a radical enzyme catalyzing the stereospecific interconversion of (S)-3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA. It consists of two subunits, HcmA and HcmB. To characterize the determinants of substrate specificity, we have analyzed the crystal structure of HCM from Aquincola tertiaricarbonis in complex with coenzyme B12 and the substrates (S)-3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA in alternative binding. When compared with the well studied structure of bacterial and mitochondrial B12-dependent methylmalonyl-CoA mutase (MCM), HCM has a highly conserved domain architecture. However, inspection of the substrate binding site identified amino acid residues not present in MCM, namely HcmA Ile(A90) and Asp(A117). Asp(A117) determines the orientation of the hydroxyl group of the acyl-CoA esters by H-bond formation, thus determining stereospecificity of catalysis. Accordingly, HcmA D117A and D117V mutations resulted in significantly increased activity toward (R)-3-hydroxybutyryl-CoA. Besides interconversion of hydroxylated acyl-CoA esters, wild-type HCM as well as HcmA I90V and I90A mutant enzymes could also isomerize pivalyl- and isovaleryl-CoA, albeit at >10 times lower rates than the favorite substrate (S)-3-hydroxybutyryl-CoA. The nonconservative mutation HcmA D117V, however, resulted in an enzyme showing high activity toward pivalyl-CoA. Structural requirements for binding and isomerization of highly branched acyl-CoA substrates such as 2-hydroxyisobutyryl- and pivalyl-CoA, possessing tertiary and quaternary carbon atoms, respectively, are discussed.

Glutamate 338 is an electrostatic facilitator of C-Co bond breakage in a dynamic/electrostatic model of catalysis by ornithine aminomutase.

How cobalamin-dependent enzymes promote C-Co homolysis to initiate radical catalysis has been debated extensively. For the pyridoxal 5'-phosphate and cobalamin-dependent enzymes lysine 5,6-aminomutase and ornithine 4,5-aminomutase (OAM), large-scale re-orientation of the cobalamin-binding domain linked to C-Co bond breakage has been proposed. In these models, substrate binding triggers dynamic sampling of the B12 -binding Rossmann domain to achieve a catalytically competent 'closed' conformational state. In 'closed' conformations of OAM, Glu338 is thought to facilitate C-Co bond breakage by close association with the cobalamin adenosyl group. We investigated this using stopped-flow continuous-wave photolysis, viscosity dependence kinetic measurements, and electron paramagnetic resonance spectroscopy of a series of Glu338 variants. We found that substrate-induced C-Co bond homolysis is compromised in Glu388 variant forms of OAM, although photolysis of the C-Co bond is not affected by the identity of residue 338. Electrostatic interactions of Glu338 with the 5'-deoxyadenosyl group of B12 potentiate C-Co bond homolysis in 'closed' conformations only; these conformations are unlocked by substrate binding. Our studies extend earlier models that identified a requirement for large-scale motion of the cobalamin domain. Our findings indicate that large-scale motion is required to pre-organize the active site by enabling transient formation of 'closed' conformations of OAM. In 'closed' conformations, Glu338 interacts with the 5'-deoxyadenosyl group of cobalamin. This interaction is required to potentiate C-Co homolysis, and is a crucial component of the approximately 10(12) rate enhancement achieved by cobalamin-dependent enzymes for C-Co bond homolysis.

A conformational sampling model for radical catalysis in pyridoxal phosphate- and cobalamin-dependent enzymes.

Cobalamin-dependent enzymes enhance the rate of C-Co bond cleavage by up to ∼10(12)-fold to generate cob(II)alamin and a transient adenosyl radical. In the case of the pyridoxal 5'-phosphate (PLP) and cobalamin-dependent enzymes lysine 5,6-aminomutase and ornithine 4,5 aminomutase (OAM), it has been proposed that a large scale domain reorientation of the cobalamin-binding domain is linked to radical catalysis. Here, OAM variants were designed to perturb the interface between the cobalamin-binding domain and the PLP-binding TIM barrel domain. Steady-state and single turnover kinetic studies of these variants, combined with pulsed electron-electron double resonance measurements of spin-labeled OAM were used to provide direct evidence for a dynamic interface between the cobalamin and PLP-binding domains. Our data suggest that following ligand binding-induced cleavage of the Lys(629)-PLP covalent bond, dynamic motion of the cobalamin-binding domain leads to conformational sampling of the available space. This supports radical catalysis through transient formation of a catalytically competent active state. Crucially, it appears that the formation of the state containing both a substrate/product radical and Co(II) does not restrict cobalamin domain motion. A similar conformational sampling mechanism has been proposed to support rapid electron transfer in a number of dynamic redox systems.

Isotope effects for deuterium transfer and mutagenesis of Tyr187 provide insight into controlled radical chemistry in adenosylcobalamin-dependent ornithine 4,5-aminomutase.

Adenosylcobalamin-dependent ornithine 4,5-aminomutase (OAM) from Clostridium sticklandii utilizes pyridoxal 5'-phosphate (PLP) to interconvert d-ornithine to 2,4-diaminopentanoate via a multistep mechanism that involves two hydrogen transfer steps. Herein, we uncover features of the OAM catalytic mechanism that differentiate it from its homologue, the more catalytically promiscuous lysine 5,6-aminomutase. Kinetic isotope effects (KIEs) with dl-ornithine-3,3,4,4,5,5-d6 revealed a diminished (D)kcat/Km of 2.5 ± 0.4 relative to a (D)kcat of 7.6 ± 0.5, suggesting slow release of the substrate from the active site. In contrast, a KIE was not observed on the rate constant associated with Co-C bond homolysis as this step is likely "gated" by the formation of the external aldimine. The role of tyrosine 187, which lies planar to the PLP pyridine ring, was also investigated via site-directed mutagenesis. The 25- and 1260-fold reduced kcat values for Y187F and Y187A, respectively, are attributed to a slower rate of external aldimine formation and a diminution of adenosylcobalamin Co-C bond homolysis. Notably, electron paramagnetic resonance studies of Y187F suggest that the integrity of the active site is maintained as cob(II)alamin and the PLP organic radical (even at lower concentrations) remain tightly exchange-coupled. Modeling of d-lysine and l-ß-lysine into the 5,6-LAM active site reveals interactions between the substrate and protein are weaker than those in OAM and fewer in number. The combined data suggest that the level of protein-substrate interactions in aminomutases not only influences substrate specificity, but also controls radical chemistry.

Paramagnetic intermediates generated by radical S-adenosylmethionine (SAM) enzymes.

A [4Fe-4S](+) cluster reduces a bound S-adenosylmethionine (SAM) molecule, cleaving it into methionine and a 5'-deoxyadenosyl radical (5'-dA(•)). This step initiates the varied chemistry catalyzed by each of the so-called radical SAM enzymes. The strongly oxidizing 5'-dA(•) is quenched by abstracting a H-atom from a target species. In some cases, this species is an exogenous molecule of substrate, for example, L-tyrosine in the [FeFe] hydrogenase maturase, HydG. In other cases, the target is a proteinaceous residue as in all the glycyl radical forming enzymes. The generation of this initial radical species and the subsequent chemistry involving downstream radical intermediates is meticulously controlled by the enzyme so as to prevent unwanted reactions. But the manner in which this control is exerted is unknown. Electron paramagnetic resonance (EPR) spectroscopy has proven to be a valuable tool used to gain insight into these mechanisms. In this Account, we summarize efforts to trap such radical intermediates in radical SAM enzymes and highlight four examples in which EPR spectroscopic results have shed significant light on the corresponding mechanism. For lysine 2,3-aminomutase, nearly each possible intermediate, from an analogue of the initial 5'-dA(•) to the product radical L-ß-lysine, has been explored. A paramagnetic intermediate observed in biotin synthase is shown to involve an auxiliary [FeS] cluster whose bridging sulfide is a co-substrate for the final step in the biosynthesis of vitamin B7. In HydG, the L-tyrosine substrate is converted in unprecedented fashion to a 4-oxidobenzyl radical on the way to generating CO and CN(-) ligands for the [FeFe] cluster of hydrogenase. And finally, EPR has confirmed a mechanistic proposal for the antibiotic resistance protein Cfr, which methylates the unactivated sp(2)-hybridized C8-carbon of an adenosine base of 23S ribosomal RNA. These four systems provide just a brief survey of the ever-growing set of radical SAM enzymes. The diverse chemistries catalyzed by these enzymes make them an intriguing target for continuing study, and EPR spectroscopy, in particular, seems ideally placed to contribute to our understanding.