A Human Vitamin B12 Trafficking Protein Uses Glutathione Transferase Activity for Processing Alkylcobalamins

Pathways for tailoring and processing vitamins into active cofactor forms exist in mammals that are unable to synthesize these cofactors de novo. A prerequisite for intracellular tailoring of alkylcobalamins entering from the circulation is removal of the alkyl group to generate an intermediate that can subsequently be converted into the active cofactor forms. MMACHC, a cytosolic cobalamin trafficking chaperone, has been shown recently to catalyze a reductive decyanation reaction when it encounters cyanocobalamin. In this study, we demonstrate that this versatile protein catalyzes an entirely different chemical reaction with alkylcobalamins using the thiolate of glutathione for nucleophilic displacement to generate cob(I)alamin and the corresponding glutathione thioether. Biologically relevant thiols, e.g. cysteine and homocysteine, cannot substitute for glutathione. The catalytic turnover numbers for the dealkylation of methylcobalamin and 5′-deoxyadenosylcobalamin by MMACHC are 11.7 ± 0.2 and 0.174 ± 0.006 h⁻¹ at 20 °C, respectively. This glutathione transferase activity of MMACHC is reminiscent of the methyltransferase chemistry catalyzed by the vitamin B₁₂-dependent methionine synthase and is impaired in the cblC group of inborn errors of cobalamin disorders.     

Mechanistic Diversity of Radical S-Adenosylmethionine (SAM)-dependent Methylation

Radical S-adenosylmethionine (SAM) enzymes use the oxidizing power of a 5′-deoxyadenosyl 5′-radical to initiate an amazing array of transformations, usually through the abstraction of a target substrate hydrogen atom. A common reaction of radical SAM (RS) enzymes is the methylation of unactivated carbon or phosphorous atoms found in numerous primary and secondary metabolites, as well as in proteins, sugars, lipids, and RNA. However, neither the chemical mechanisms by which these unactivated atoms obtain methyl groups nor the actual methyl donors are conserved. In fact, RS methylases have been grouped into three classes based on protein architecture, cofactor requirement, and predicted mechanism of catalysis. Class A methylases use two cysteine residues to methylate sp²-hybridized carbon centers. Class B methylases require a cobalamin cofactor to methylate both sp²-hybridized and sp³-hybridized carbon centers as well as phosphinate phosphorous atoms. Class C methylases share significant sequence homology with the RS enzyme, HemN, and may bind two SAM molecules simultaneously to methylate sp²-hybridized carbon centers. Lastly, we describe a new class of recently discovered RS methylases. These Class D methylases, unlike Class A, B, and C enzymes, which use SAM as the source of the donated methyl carbon, are proposed to methylate sp²-hybridized carbon centers using methylenetetrahydrofolate as the source of the appended methyl carbon.     

S-adenosylmethionine-dependent enzyme activation

S-Adenosylmethionine (SAM)-dependent activations of pyruvate formate-lyase, lysine 2,3-aminomutase and cobalamin-dependent methionine synthase are discussed. In each case, cleavage of SAM is accompanied by the formation of a catalytically active enzyme. The chemistry of activation of these three enzymes falls into three distinct classes: generation of an essential enzyme radical (pyruvate formate-lyase), formation of a catalytically active 5'-deoxyadenosyl radical (lysine 2,3-aminomutase) and reductive methylation to form a required methylcobalamin complex (methionine synthase).     

Tumor uptake studies of S-adenosyl-l-[methyl-11C]methionine and l-[methyl-11C]methionine

Tumor accumulation of S-adenosyl-l-[methyl-¹¹C]methionine ([¹¹C]SAM) was investigated in mice bearing mammary carcinoma (FM3A) and in rats bearing ascitic hepatoma (AH109A). After injection of [¹¹C]SAM the blood clearance of ¹¹C radioactivity was rapid. The ¹¹C level was relatively high in both tumors. The uptake ratios of tumor to organ increased with time in several organs, especially in brain and muscle. In FM3A tumor tissue the ¹¹C was incorporated with time into the acid-precipitable fraction and 38% of the ¹¹C was detected in this fraction at 60 min after injection. This fraction reflects the amount of ¹¹C-methyl group transferred into macromolecules in tumor tissue. In AH109A-bearing rats the metabolisms of [¹¹C]SAM and l-[methyl-¹¹C]methionine ([¹¹C]Met), in vivo precursor of SAM, were compared. Tumor uptake of [¹¹C]SAM was about two thirds of that of [¹¹C]Met at 20 min after injection. At this time, for the [¹¹C]SAM 27 and 8% of the ¹¹C in the AH109A tissue were detected in the acid-precipitable and the lipid fractions, respectively. The corresponding figures for [¹¹C]Met were 61% and 2%. In the liver considerable amounts of ¹¹C were observed in the lipid fraction for both tracers.These results show that [¹¹C]SAM has potential as a tracer for tumor localization with positron emission tomography (PET) and suggest that in tumor studies combining [¹¹C]Met and PET, it should be taken into account that the ¹¹C-labeled methyl group of [¹¹C]Met is not only incorporated into protein but also other macromolecules and lipids via [¹¹C]SAM.     

Structural insights into auxiliary cofactor usage by radical S-adenosylmethionine enzymes

Radical S-adenosylmethionine (SAM) enzymes use a common catalytic core for diverse transformations. While all radical SAM enzymes bind a Fe₄S₄ cluster via a characteristic tri-cysteine motif, many bind additional metal cofactors. Recently reported structures of radical SAM enzymes that use methylcobalamin or additional iron-sulfur clusters as cosubstrates show that these auxiliary units are anchored by N- and C-terminal domains that vary significantly in size and topology. Despite this architectural diversity, all use a common surface for auxiliary cofactor docking. In the sulfur insertion and metallocofactor assembly systems evaluated here, interaction with iron-sulfur cluster assembly proteins or downstream scaffold proteins is an important component of catalysis. Structures of these complexes represent important new frontiers in structural analysis of radical SAM enzymes.     

Dihydroflavin-driven Adenosylation of 4-Coordinate Co(II) Corrinoids: ARE COBALAMIN REDUCTASES ENZYMES OR ELECTRON TRANSFER PROTEINS?*

The identity of the source of the biological reductant needed to convert cobalamin to its biologically active form adenosylcobalamin has remained elusive. Here we show that free or protein-bound dihydroflavins can serve as the reductant of Co²⁺Cbl bound in the active site of PduO-type ATP-dependent corrinoid adenosyltransferase enzymes. Free dihydroflavins (dihydroriboflavin, FMNH₂, and FADH₂) effectively drove the adenosylation of Co²⁺Cbl by the human and bacterial PduO-type enzymes at very low concentrations (1 μm). These data show that adenosyltransferase enzymes lower the thermodynamic barrier of the Co²⁺ → Co⁺ reduction needed for the formation of the unique organometalic Co–C bond of adenosylcobalamin. Collectively, our in vivo and in vitro data suggest that cobalamin reductases identified thus far are most likely electron transfer proteins, not enzymes.     

5-methyltetrahydrofolate homocysteine cobalamin methyltransferase in human bone marrow and its relationship to pernicious anemia

Dialyzed extracts from human bone marrow catalyze [5-¹⁴C]methyltetrahydrofolate homocysteine transmethylation at slow but significant rates which can be detected by using substrate with a very high specific radioactivity. Enzymatic activity is associated with nucleated marrow cells rather than mature, nondividing erythrocytes. Extract transmethylase activities in 15 marrow specimens from patients without B-12 deficiency ranged from 157–1020 pmoles of [Me-¹⁴C]methionine formed/hr/10⁷ nucleated cells. Catalysis is dependent on S-adenosyl-l-methionine and a flavin-reducing system, typical for the presence of a cobalamin (B-12) methyltransferase. No in vitro requirement for exogenous B-12 was observed except for the marrow extracts from two patients known to be B-12 deficient. One of these extracts was markedly stimulated by methyl-B-12 indicative that mostly apomethyltransferase was present. These tracer assays with cell-free extracts provide the first direct evidence that human bone marrow contains B-12 methyltransferase; they also afford further evidence for a 5-methyltetrahydrofolate trap in B-12 deficiency with its associated megaloblastic anemia. In addition, we have observed that in normal peripheral blood leukocytes the mononuclear fraction contains 10–30 times as much B-12 methyltransferase per nucleated cell as the polymorphonuclear granulocyte fraction.     

Identification of a Unique Radical S-Adenosylmethionine Methylase Likely Involved in Methanopterin Biosynthesis in Methanocaldococcus jannaschii

Methanopterin (MPT) and its analogs are coenzymes required for methanogenesis and methylotrophy in specialized microorganisms. The methyl groups at C-7 and C-9 of the pterin ring distinguish MPT from all other pterin-containing natural products. However, the enzyme(s) responsible for the addition of these methyl groups has yet to be identified. Here we demonstrate that a putative radical S-adenosyl-l-methionine (SAM) enzyme superfamily member encoded by the MJ0619 gene in the methanogen Methanocaldococcus jannaschii is likely this missing methylase. When MJ0619 was heterologously expressed in Escherichia coli, various methylated pterins were detected, consistent with MJ0619 catalyzing methylation at C-7 and C-9 of 7,8-dihydro-6-hydroxymethylpterin, a common intermediate in both folate and MPT biosynthesis. Site-directed mutagenesis of Cys77 present in the first of two canonical radical SAM CX₃CX₂C motifs present in MJ0619 did not inhibit C-7 methylation, while mutation of Cys102, found in the other radical SAM amino acid motif, resulted in the loss of C-7 methylation, suggesting that the first motif could be involved in C-9 methylation, while the second motif is required for C-7 methylation. Further experiments demonstrated that the C-7 methyl group is not derived from methionine and that methylation does not require cobalamin. When E. coli cells expressing MJ0619 were grown with deuterium-labeled acetate as the sole carbon source, the resulting methyl group on the pterin was predominantly labeled with three deuteriums. Based on these results, we propose that this archaeal radical SAM methylase employs a previously uncharacterized mechanism for methylation, using methylenetetrahydrofolate as a methyl group donor.     

The Radical S-Adenosyl-l-methionine Enzyme QhpD Catalyzes Sequential Formation of Intra-protein Sulfur-to-Methylene Carbon Thioether Bonds

The bacterial enzyme designated QhpD belongs to the radical S-adenosyl-l-methionine (SAM) superfamily of enzymes and participates in the post-translational processing of quinohemoprotein amine dehydrogenase. QhpD is essential for the formation of intra-protein thioether bonds within the small subunit (maturated QhpC) of quinohemoprotein amine dehydrogenase. We overproduced QhpD from Paracoccus denitrificans as a stable complex with its substrate QhpC, carrying the 28-residue leader peptide that is essential for the complex formation. Absorption and electron paramagnetic resonance spectra together with the analyses of iron and sulfur contents suggested the presence of multiple (likely three) [4Fe-4S] clusters in the purified and reconstituted QhpD. In the presence of a reducing agent (sodium dithionite), QhpD catalyzed the multiple-turnover reaction of reductive cleavage of SAM into methionine and 5′-deoxyadenosine and also the single-turnover reaction of intra-protein sulfur-to-methylene carbon thioether bond formation in QhpC bound to QhpD, producing a multiknotted structure of the polypeptide chain. Homology modeling and mutagenic analysis revealed several conserved residues indispensable for both in vivo and in vitro activities of QhpD. Our findings uncover another challenging reaction catalyzed by a radical SAM enzyme acting on a ribosomally translated protein substrate.     

Function of methylcobalamin: coenzyme M methyltransferase isoenzyme II in Methanosarcina barkeri

Methanosarcina barkeri was recently shown to contain two cytoplasmic isoenzymes of methylcobalamin: coenzyme M methyltransferase (methyltransferase 2). Isoenzyme I predominated in methanol-grown cells and isoenzyme II in acetate-grown cells. It was therefore suggested that isoenzyme I functions in methanogenesis from methanol and isoenzyme II in methanogenesis from acetate. We report here that cells of M. barkeri grown on trimethylamine, H₂/CO₂, or acetate contain mainly isoenzyme II. These cells were found to have in common that they can catalyze the formation of methane from trimethylamine and H₂, whereas only acetate-grown cells can mediate the formation of methane from acetate. Methanol-grown cells, which contained only low concentrations of isoenzyme II, were unable to mediate the formation of methane from both trimethylamine and acetate. These and other results suggest that isoenzyme II (i) is employed for methane formation from trimethylamine rather than from acetate, (ii) is constitutively expressed rather than trimethylamine-induced, and (iii) is repressed by methanol. The constitutive expression of isoenzyme II in acetate-grown M. barkeri can explain its presence in these cells. The N-terminal amino acid sequences of isoenzyme I and isoenzyme II were analyzed and found to be only 55% similar.Abbreviations H-S-CoM coenzyme M or 2-mercaptoethane-sulfonate - CH₃-S-CoM methyl-coenzyme M or 2(methylthio)-ethanesulfonate - [Co] cobalamin - CH₃-[Co] methylcobalamin - H₄MPT tetrahydromethanopterin - CH₃-H₄MPT N ⁵-methyltetrahydromethanopterin - MT₁ methyltransferase 1 or methanol: 5-hydroxybenzimidazolyl cobamide methyltransferase - MT₂ methyltransferase 2 or methylcobalamin: coenzyme M methyltransferase - Mops morpholinopropanesulfonate - 1 U = 1 mol/min     

A new mode of B12 binding and the direct participation of a potassium ion in enzyme catalysis: X-ray structure of diol dehydratase

Background: Diol dehydratase is an enzyme that catalyzes the adenosylcobalamin (coenzyme B₁₂) dependent conversion of 1,2-diols to the corresponding aldehydes. The reaction initiated by homolytic cleavage of the cobalt–carbon bond of the coenzyme proceeds by a radical mechanism. The enzyme is an α₂β₂γ₂ heterooligomer and has an absolute requirement for a potassium ion for catalytic activity. The crystal structure analysis of a diol dehydratase–cyanocobalamin complex was carried out in order to help understand the mechanism of action of this enzyme.Results: The three-dimensional structure of diol dehydratase in complex with cyanocobalamin was determined at 2.2 Å resolution. The enzyme exists as a dimer of heterotrimers (α β γ)₂. The cobalamin molecule is bound between the α and β subunits in the ‘base-on’ mode, that is, 5,6-dimethylbenzimidazole of the nucleotide moiety coordinates to the cobalt atom in the lower axial position. The α subunit includes a (β/α)₈ barrel. The substrate, 1,2-propanediol, and an essential potassium ion are deeply buried inside the barrel. The two hydroxyl groups of the substrate coordinate directly to the potassium ion.Conclusions: This is the first crystallographic indication of the ‘base-on’ mode of cobalamin binding. An unusually long cobalt–base bond seems to favor homolytic cleavage of the cobalt–carbon bond and therefore to favor radical enzyme catalysis. Reactive radical intermediates can be protected from side reactions by spatial isolation inside the barrel. On the basis of unique direct interactions between the potassium ion and the two hydroxyl groups of the substrate, direct participation of a potassium ion in enzyme catalysis is strongly suggested.     

Cytosine-to-Uracil Deamination by SssI DNA Methyltransferase

The prokaryotic DNA(cytosine-5)methyltransferase M.SssI shares the specificity of eukaryotic DNA methyltransferases (CG) and is an important model and experimental tool in the study of eukaryotic DNA methylation. Previously, M.SssI was shown to be able to catalyze deamination of the target cytosine to uracil if the methyl donor S-adenosyl-methionine (SAM) was missing from the reaction. To test whether this side-activity of the enzyme can be used to distinguish between unmethylated and C5-methylated cytosines in CG dinucleotides, we re-investigated, using a sensitive genetic reversion assay, the cytosine deaminase activity of M.SssI. Confirming previous results we showed that M.SssI can deaminate cytosine to uracil in a slow reaction in the absence of SAM and that the rate of this reaction can be increased by the SAM analogue 5’-amino-5’-deoxyadenosine. We could not detect M.SssI-catalyzed deamination of C5-methylcytosine (^m5C). We found conditions where the rate of M.SssI mediated C-to-U deamination was at least 100-fold higher than the rate of ^m5C-to-T conversion. Although this difference in reactivities suggests that the enzyme could be used to identify C5-methylated cytosines in the epigenetically important CG dinucleotides, the rate of M.SssI mediated cytosine deamination is too low to become an enzymatic alternative to the bisulfite reaction. Amino acid replacements in the presumed SAM binding pocket of M.SssI (F17S and G19D) resulted in greatly reduced methyltransferase activity. The G19D variant showed cytosine deaminase activity in E. coli, at physiological SAM concentrations. Interestingly, the C-to-U deaminase activity was also detectable in an E. coli ung ⁺ host proficient in uracil excision repair.     

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 ∼10¹²-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 Lys⁶²⁹-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.     

Chlamydomonas reinhardtii thermal tolerance enhancement mediated by a mutualistic interaction with vitamin B12-producing bacteria

Temperature is one of the most important environmental factors affecting the growth and survival of microorganisms and in light of current global patterns is of particular interest. Here, we highlight studies revealing how vitamin B₁₂ (cobalamin)-producing bacteria increase the fitness of the unicellular alga Chlamydomonas reinhardtii following an increase in environmental temperature. Heat stress represses C. reinhardtii cobalamin-independent methionine synthase (METE) gene expression coinciding with a reduction in METE-mediated methionine synthase activity, chlorosis and cell death during heat stress. However, in the presence of cobalamin-producing bacteria or exogenous cobalamin amendments C. reinhardtii cobalamin-dependent methionine synthase METH-mediated methionine biosynthesis is functional at temperatures that result in C. reinhardtii death in the absence of cobalamin. Artificial microRNA silencing of C. reinhardtii METH expression leads to nearly complete loss of cobalamin-mediated enhancement of thermal tolerance. This suggests that methionine biosynthesis is an essential cellular mechanism for adaptation by C. reinhardtii to thermal stress. Increased fitness advantage of METH under environmentally stressful conditions could explain the selective pressure for retaining the METH gene in algae and the apparent independent loss of the METE gene in various algal species. Our results show that how an organism acclimates to a change in its abiotic environment depends critically on co-occurring species, the nature of that interaction, and how those species interactions evolve.     

5-Methyltetrahydrofolate aromatic alkylamine N-methyltransferase: an artefact of 5,10-methylenetetrahydrofolate reductase activity.

A previously reported stimulation of brain 5-methyltetrahydrofolate (5-MeH₄-folate) N-methyltransferase by FAD and methylcobalamin (MeB₁₂ is attributed to their roles as nonspecific electron acceptors. Evidence is presented that the catalyst involved is not an aromatic alkylamine methyltransferase, but the widely distributed enzyme, 5,10-methyleneH₄-folate reductase. In the presence of an electron acceptor it catalyzes the oxidation of [5-¹⁴C]MeH₄-folate to [5,10-¹⁴C]methyleneH₄-folate which equilibrates to yield dimedone reactive H¹⁴CHO. The material being measured when incubation systems containing β-phenylethylamine or tryptamine are extracted with tolueneisoamyl alcohol is a condensation product of the H¹⁴CHO and the aromatic alkylamine. The aromatic alkylamine is not a co-substrate in the enzymic oxidation mechanism. It is required to react nonenzymically with reductase formed H¹⁴CHO and render it extractable. Our failure and that recently of others to detect significant N-methylation using [5-¹⁴C]MeH₄-folate as a Me group donor make the existence of a folate-biogenic amine methyltransferase seem highly improbable.     

In vitro phosphinate methylation by PhpK from Kitasatospora phosalacinea

Radical S-adenosyl-L-methionine, cobalamin-dependent methyltransferases have been proposed to catalyze the methylations of unreactive carbon or phosphorus atoms in antibiotic biosynthetic pathways. To date, none of these enzymes has been purified or shown to be active in vitro. Here we demonstrate the activity of the P-methyltransferase enzyme, PhpK, from the phosalacine producer Kitasatospora phosalacinea. PhpK catalyzes the transfer of a methyl group from methylcobalamin to 2-acetylamino-4-hydroxyphosphinylbutanoate (N-acetyldemethylphosphinothricin) to form 2-acetylamino-4-hydroxymethylphosphinylbutanoate (N-acetylphosphinothricin). This transformation gives rise to the only carbon-phosphorus-carbon linkage known to occur in nature.     

Adenosylation reactions catalyzed by the radical S-adenosylmethionine superfamily enzymes

The radical S-adenosylmethionine (SAM) superfamily enzymes reductively cleave SAM to produce a highly reactive 5ˊ-deoxyadenosyl (dAdo) radical, which in most cases abstracts a hydrogen from the substrate and initiates highly diverse reactions. In rare cases, the dAdo radical can add to a sp² carbon to result in the production an adenosylated product. These radical SAM-dependent adenosylation reactions are present in natural product biosynthetic pathways and can be achieved by using unnatural substrate analogs containing olefin or aryl moieties. This Opinion provides a focused perspective on this emerging type of biochemistry and discusses its potential use in bioengineering and biocatalysis.     

Guanidinoacetate Is More Effective than Creatine at Enhancing Tissue Creatine Stores while Consequently Limiting Methionine Availability in Yucatan Miniature Pigs

Creatine (Cr) is an important high-energy phosphate buffer in tissues with a high energy demand such as muscle and brain and is consequently a highly consumed nutritional supplement. Creatine is synthesized via the S-adenosylmethionine (SAM) dependent methylation of guanidinoacetate (GAA) which is not regulated by a feedback mechanism. The first objective of this study was to determine the effectiveness of GAA at increasing tissue Cr stores. Because SAM is required for other methylation reactions, we also wanted to determine whether an increased creatine synthesis would lead to a lower availability of methyl groups for other methylated products. Three month-old pigs (n = 18) were fed control, GAA- or Cr-supplemented diets twice daily. On day 18 or 19, anesthesia was induced 1–3 hours post feeding and a bolus of [methyl-³H]methionine was intravenously infused. After 30 minutes, the liver was analyzed for methyl-³H incorporation into protein, Cr, phosphatidylcholine (PC) and DNA. Although both Cr and GAA led to higher hepatic Cr concentration, only supplementation with GAA led to higher levels of muscle Cr (P < 0.05). Only GAA supplementation resulted in lower methyl-³H incorporation into PC and protein as well as lower hepatic SAM concentration compared to the controls, suggesting that Cr synthesis resulted in a limited methyl supply for PC and protein synthesis (P < 0.05). Although GAA is more effective than Cr at supporting muscle Cr accretion, further research should be conducted into the long term consequences of a limited methyl supply and its effects on protein and PC homeostasis.     

Discovery and Characterization of BlsE,a Radical S-Adenosyl-L-methionine Decarboxylase Involved in the Blasticidin S Biosynthetic Pathway

BlsE, a predicted radical S-adenosyl-L-methionine (SAM) protein, was anaerobically purified and reconstituted in vitro to study its function in the blasticidin S biosynthetic pathway. The putative role of BlsE was elucidated based on bioinformatics analysis, genetic inactivation and biochemical characterization. Biochemical results showed that BlsE is a SAM-dependent radical enzyme that utilizes cytosylglucuronic acid, the accumulated intermediate metabolite in blsE mutant, as substrate and catalyzes decarboxylation at the C5 position of the glucoside residue to yield cytosylarabinopyranose. Additionally, we report the purification and reconstitution of BlsE, characterization of its [4Fe–4S] cluster using UV-vis and electron paramagnetic resonance (EPR) spectroscopic analysis, and investigation of the ability of flavodoxin (Fld), flavodoxin reductase (Fpr) and NADPH to reduce the [4Fe–4S]²⁺ cluster. Mutagenesis studies demonstrated that Cys₃₁, Cys_35, Cys₃₈ in the C×××C×MC motif and Gly₇₃, Gly₇₄, Glu₇₅, Pro₇₆ in the GGEP motif were crucial amino acids for BlsE activity while mutation of Met₃₇ had little effect on its function. Our results indicate that BlsE represents a typical [4Fe–4S]-containing radical SAM enzyme and it catalyzes decarboxylation in blasticidin S biosynthesis.     

Conversion and Distribution of Cobalamin in Euglena gracilis z, with Special Reference to Its Location and Probable Function within Chloroplasts

Cobalamin is essentially required for growth by Euglena gracilis and shown to be converted to coenzyme forms promptly after feeding cyanocobalamin. Concentrations of coenzymes, methylcobalamin, and 5′-deoxyadenosylcobalamin, reached about 1 femtomole/10⁶ cells 2 hours after feeding cyanocobalamin to cobalamin-limited cells. Cobalamins all were bound to proteins in Euglena cells and located in subcellular fractions of chloroplasts, mitochondria, microsomes, and cytosol. Incorporated cobalamin into chloroplasts was localized in thylakoids. Methylcobalamin existed in chloroplasts, mitochondria, and cytosol, while 5′-deoxyadenosylcobalamin was in mitochondria and the cytosol, 2 h after feeding cyanocobalamin to Euglena cells. Quantitative alterations of methylcobalamin and 5′-deoxyadenosylcobalamin in chloroplasts suggest their important functions as coenzymes in this organelle. The occurrence of functional cobalamins in chloroplasts has not been reported in other photosynthetic eukaryotes.