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The dietary intake of vitamin B12 among unsupplemented vegans is notably lower compared to both vegetarians and omnivores. Prolonged low intakes of vitamin B12, such as seen in those adhering to a vegan diet, lead to physiological deficiency of vitamin B12 and an elevated risk of B12‐related morbidity. However, while serum B12 serves as a conventional biomarker for assessing B12 status, its utility is limited given its sensitivity and specificity in ascribing physiological deficiency of B12 and the functional vitamin B12 status of those adhering to vegan diets is unclear. We conducted a systematic review and meta‐analysis using data based on the full panel of biomarkers of vitamin B12 status to test whether adherence to a vegan diet is associated with an elevated risk of functional vitamin B12 deficiency compared to vegetarian or omnivorous diets. In addition, subgroup analysis was carried out to look at the effect of vitamin B12 supplement use on B12 status among vegans. Our search identified 4002 records, of which 19 studies met the inclusion criteria for the systematic review and 17 studies were taken forward for the meta‐analysis. Meta‐analysis results revealed significantly lower serum B12, pmol/ (−0.72 [−1.26, −0.18]; p = 0.01) and elevated total homocysteine, μmol/L (tHcy) (0.57 [0.26, 0.89]; p < 0.001) concentrations, alongside elevated methylmalonic acid, nmol/L (MMA) (0.28 [−0.01, 0.57]; p = 0.06) and lower holotranscobalamin, pmol/ (HoloTC) (−0.42 [−0.91, 0.07]; p = 0.09) levels among vegan adults compared to omnivores, indicating increased functional B12 deficiency in addition to low vitamin B12 status in vegan adults. There were no differences between vegans and vegetarians in HoloTC (0.04 [−0.28, 0.35]; p = 0.814) or MMA (−0.05 [−0.29, 0.20]; p = 0.708), but differences were found in serum B12 (−0.25 [−0.40, −0.10]; p = 0.001) and for tHcy (0.24 [0.09, 0.39]; p = 0.002) concentrations. Subgroup analyses indicated that the use of vitamin B12 supplements among vegans contributes to significant improvements in all biomarker concentrations compared to their unsupplemented counterparts. Our findings underscore the need for improved strategies to redress poor vitamin B12 status with appropriate B12 supplementation use among those adhering to vegetarian and vegan diets. [ABSTRACT FROM AUTHOR]

Coenzyme B12 (AdoCbl; 5′‐deoxy‐5′‐adenosylcobalamin), the quintessential biological organometallic radical catalyst, has a formerly unanticipated, yet extensive, role in photoregulation in bacteria. The light‐responsive cobalt‐corrin AdoCbl performs this nonenzymatic role by facilitating the assembly of CarH photoreceptors into DNA‐binding tetramers in the dark, suppressing gene expression. Conversely, exposure to light triggers the decomposition of this AdoCbl‐bound complex by a still elusive photochemical mechanism, activating gene expression. Here, we have examined AdoRhbl, the non‐natural rhodium analogue of AdoCbl, as a photostable isostructural surrogate for AdoCbl. We show that AdoRhbl closely emulates AdoCbl in its uptake by bacterial cells and structural functionality as a regulatory ligand for CarH tetramerization, DNA binding, and repressor activity. Remarkably, we find AdoRhbl is photostable even when bound "base‐off/His‐on" to CarH in vitro and in vivo. Thus, AdoRhbl, an antivitamin B12, also represents an unprecedented anti‐photoregulatory ligand, opening a pathway to precisely target biomimetic inhibition of AdoCbl‐based photoregulation, with new possibilities for selective antibacterial applications. Computational biomolecular analysis of AdoRhbl binding to CarH yields detailed structural insights into this complex, which suggest that the adenosyl group of photoexcited AdoCbl bound to CarH may specifically undergo a concerted non‐radical syn‐1,2‐elimination mechanism, an aspect not previously considered for this photoreceptor. [ABSTRACT FROM AUTHOR]

Bacterial extracellular vesicles (BEVs) contribute to stress responses, quorum sensing, biofilm formation and interspecies and interkingdom communication. However, the factors that regulate their release and heterogeneity are not well understood. We set out to investigate these factors in the common gut commensal Bacteroides thetaiotaomicron by studying BEV release throughout their growth cycle. Utilising a range of methods, we demonstrate that vesicles released at different stages of growth have significantly different composition, with early vesicles enriched in specifically released outer membrane vesicles (OMVs) containing a larger proportion of lipoproteins, while late phase BEVs primarily contain lytic vesicles with enrichment of cytoplasmic proteins. Furthermore, we demonstrate that lipoproteins containing a negatively charged signal peptide are preferentially incorporated in OMVs. We use this observation to predict all Bacteroides thetaiotaomicron OMV enriched lipoproteins and analyse their function. Overall, our findings highlight the need to understand media composition and BEV release dynamics prior to functional characterisation and define the theoretical functional capacity of Bacteroides thetaiotaomicron OMVs. [ABSTRACT FROM AUTHOR]

The author comments on the diet and health Open Innovation Research Club (OIRC) innovation hubs designed to identify solutions to the key diet, health and nutrition challenges facing the United Kingdom. Cited are how the innovation hubs will reinvigorate and strengthen nutrition research, how the hubs are structured to engage the food industry in recognition of their role in progressing shared challenges, and the innovative opportunity the hubs provide to advance nutrition research.

The natural vitamin B12‐derivatives are intriguing complexes of cobalt that entrap the metal within the strikingly skewed and ring‐contracted corrin ligand. Here, we describe the synthesis of the Rh(III)‐corrin acetylrhodibalamin (AcRhbl) from biotechnologically produced metal‐free hydrogenobyric acid and analyze the effect of the replacement of the cobalt‐center of the organometallic vitamin B12‐derivative acetylcobalamin (AcCbl) with its group‐IX homologue rhodium, to give AcRhbl. The structures of AcCbl and AcRhbl were thoroughly analyzed in aqueous solution, in crystals and by in silico methods, in order to gain detailed insights into the structural adaptations to the two homologous metals. Indeed, the common, nucleotide‐appended corrin‐ligand in these two metal corrins features extensive structural similarity. Thus, the rhodium‐corrin AcRhbl joins the small group of B12‐mimics classified as 'antivitamins B12', isostructural metal analogues of the natural cobalt‐corrins that hold significant potential in biological and biomedical applications as selective inhibitors of key cellular processes. [ABSTRACT FROM AUTHOR]

Summary: Cobalamin (vitamin B12) is a cofactor for essential metabolic reactions in multiple eukaryotic taxa, including major primary producers such as algae, and yet only prokaryotes can produce it. Many bacteria can colonize the algal phycosphere, forming stable communities that gain preferential access to photosynthate and in return provide compounds such as B12. Extended coexistence can then drive gene loss, leading to greater algal–bacterial interdependence. In this study, we investigate how a recently evolved B12‐dependent strain of Chlamydomonas reinhardtii, metE7, forms a mutualism with certain bacteria, including the rhizobium Mesorhizobium loti and even a strain of the gut bacterium E. coli engineered to produce cobalamin. Although metE7 was supported by B12 producers, its growth in co‐culture was slower than the B12‐independent wild‐type, suggesting that high bacterial B12 provision may be necessary to favour B12 auxotrophs and their evolution. Moreover, we found that an E. coli strain that releases more B12 makes a better mutualistic partner, and although this trait may be more costly in isolation, greater B12 release provided an advantage in co‐cultures. We hypothesize that, given the right conditions, bacteria that release more B12 may be selected for, particularly if they form close interactions with B12‐dependent algae. [ABSTRACT FROM AUTHOR]

A simple model for the B12 ‐riboswitch regulatory network in Escherichia coli is first described and the same analysis is applied when changing the strain to Salmonella enterica. Model validation is undertaken by linking the dynamics of the riboswitch model to bacterial growth and comparing the results obtained with in vivo experimental measurements. Measurements of bacterial growth are relatively straightforward to obtain experimentally, but experimental measurements relating to the operation of the riboswitch are more difficult. Using the validated model, sliding mode observer design methods are used to estimate BtuB given measurements of the concentration of vitamin B12. The sliding mode approach is selected because of its inherent robustness properties as well as for the ease of implementation. Validation of the estimates of BtuB produced by the observer is undertaken by comparing the BtuB and vitamin B12 concentrations estimated from the observer with green fluorescent protein production and the concentration of vitamin B12 obtained experimentally. These experimental results also provide further validation of the underpinning mathematical model. The results establish that using a sliding mode observer as a soft sensor is a useful approach to explore the operation of a vitamin B12 riboswitch given measurements of the concentration of vitamin B12. [ABSTRACT FROM AUTHOR]

The (formal) replacement of Co in cobalamin (Cbl) by NiII generates nibalamin (Nibl), a new transition‐metal analogue of vitamin B12. Described here is Nibl, synthesized by incorporation of a NiII ion into the metal‐free B12 ligand hydrogenobalamin (Hbl), itself prepared from hydrogenobyric acid (Hby). The related NiII corrin nibyric acid (Niby) was similarly synthesized from Hby, the metal‐free cobyric acid ligand. The solution structures of Hbl, and Niby and Nibl, were characterized by spectroscopic studies. Hbl features two inner protons bound at N2 and N4 of the corrin ligand, as discovered in Hby. X‐ray analysis of Niby shows the structural adaptation of the corrin ligand to NiII ions and the coordination behavior of NiII. The diamagnetic Niby and Nibl, and corresponding isoelectronic CoI corrins, were deduced to be isostructural. Nibl is a structural mimic of four‐coordinate base‐off Cbls, as verified by its ability to act as a strong inhibitor of bacterial adenosyltransferase. [ABSTRACT FROM AUTHOR]

Replacing the central cobalt ion of vitamin B12 by other metals has been a long‐held aspiration within the B12‐field. Herein, we describe the synthesis from hydrogenobyric acid of zincobyric acid (Znby) and zincobalamin (Znbl), the Zn‐analogues of the natural cobalt‐corrins cobyric acid and vitamin B12, respectively. The solution structures of Znby and Znbl were studied by NMR‐spectroscopy. Single crystals of Znby were produced, providing the first X‐ray crystallographic structure of a zinc corrin. The structures of Znby and of computationally generated Znbl were found to resemble the corresponding CoII‐corrins, making such Zn‐corrins potentially useful for investigations of B12‐dependent processes. The singlet excited state of Znby had a short life‐time, limited by rapid intersystem crossing to the triplet state. Znby allowed the unprecedented observation of a corrin triplet (ET=190 kJ mol−1) and was found to be an excellent photo‐sensitizer for 1O2 (ΦΔ=0.70). [ABSTRACT FROM AUTHOR]

Copyright of Angewandte Chemie is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The B12 cofactors instill a natural curiosity regarding the primordial selection and evolution of their corrin ligand. Surprisingly, this important natural macrocycle has evaded molecular scrutiny, and its specific role in predisposing the incarcerated cobalt ion for organometallic catalysis has remained obscure. Herein, we report the biosynthesis of the cobalt‐free B12 corrin moiety, hydrogenobyric acid (Hby), a compound crafted through pathway redesign. Detailed insights from single‐crystal X‐ray and solution structures of Hby have revealed a distorted helical cavity, redefining the pattern for binding cobalt ions. Consequently, the corrin ligand coordinates cobalt ions in desymmetrized "entatic" states, thereby promoting the activation of B12‐cofactors for their challenging chemical transitions. The availability of Hby also provides a route to the synthesis of transition metal analogues of B12. [ABSTRACT FROM AUTHOR]

Summary: Haem is an essential cofactor in central metabolic pathways in the vast majority of living systems. Prokaryotes acquire haem via haem biosynthesis pathways, and some also utilize haem uptake systems, yet it remains unclear how they balance haem requirements with the paradox that free haem is toxic. Here, using the model pathogen Staphylococcus aureus, we report that IsdG, one of two haem oxygenase enzymes in the haem uptake system, inhibits the formation of haem via the internal haem biosynthesis route. More specifically, we show that IsdG decreases the activity of ferrochelatase and that the two proteins interact both in vitro and in vivo. Further, a bioinformatics analysis reveals that a significant number of haem biosynthesis pathway containing organisms possess an IsdG‐homologue and that those with both biosynthesis and uptake systems have at least two haem oxygenases. We conclude that IsdG‐like proteins control intracellular haem levels by coupling the two pathways. IsdG is thus a target for the treatment of S. aureusinfections. [ABSTRACT FROM AUTHOR]

The sulfate-reducing bacteria of the Desulfovibrio genus make three distinct modified tetrapyrroles, haem, sirohaem and adenosylcobamide, where sirohydrochlorin acts as the last common biosynthetic intermediate along the branched tetrapyrrole pathway. Intriguingly, D. vulgaris encodes two sirohydrochlorin chelatases, CbiKP and CbiKC, that insert cobalt/iron into the tetrapyrrole macrocycle but are thought to be distinctly located in the periplasm and cytoplasm respectively. Fusing GFP onto the C-terminus of CbiKP confirmed that the protein is transported to the periplasm. The structure-function relationship of CbiKP was studied by constructing eleven site-directed mutants and determining their chelatase activities, oligomeric status and haem binding abilities. Residues His154 and His216 were identified as essential for metal-chelation of sirohydrochlorin. The tetrameric form of the protein is stabilized by Arg54 and Glu76, which form hydrogen bonds between two subunits. His96 is responsible for the binding of two haem groups within the main central cavity of the tetramer. Unexpectedly, CbiKP is shown to bind two additional haem groups through interaction with His103. Thus, although still retaining cobaltochelatase activity, the presence of His96 and His103 in CbiKP, which are absent from all other known bacterial cobaltochelatases, has evolved CbiKP a new function as a haem binding protein permitting it to act as a potential haem chaperone or transporter. [ABSTRACT FROM AUTHOR]

Copyright of Angewandte Chemie is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

B12 is unique among the vitamins as it is biosynthesized only by certain prokaryotes. The complexity of its synthesis relates to its distinctive cobalt corrin structure, which is essential for B12 biochemistry and renders coenzyme B12 (AdoCbl) so intriguingly suitable for enzymatic radical reactions. However, why is cobalt so fit for its role in B12-dependent enzymes? To address this question, we considered the substitution of cobalt in AdoCbl with rhodium to generate the rhodium analogue 5′-deoxy-5′-adenosylrhodibalamin (AdoRbl). AdoRbl was prepared by de novo total synthesis involving both biological and chemical steps. AdoRbl was found to be inactive in vivo in microbial bioassays for methionine synthase and acted as an in vitro inhibitor of an AdoCbl-dependent diol dehydratase. Solution NMR studies of AdoRbl revealed a structure similar to that of AdoCbl. However, the crystal structure of AdoRbl revealed a conspicuously better fit of the corrin ligand for RhIII than for CoIII, challenging the current views concerning the evolution of corrins. [ABSTRACT FROM AUTHOR]

Haem is a life supporting molecule that is ubiquitous in all major kingdoms. In S taphylococcus aureus, the importance of haem is highlighted by the presence of systems both for the exogenous acquisition and endogenous synthesis of this prosthetic group. In this work, we show that in S . aureus the formation of haem involves the conversion of coproporphyrinogen III into coproporphyrin III by coproporphyrin synthase HemY, insertion of iron into coproporphyrin III via ferrochelatase HemH, and oxidative decarboxylation of Fe-coproporphyrin III into protohaem IX by Fe-coproporphyrin oxidase/dehydrogenase HemQ. Together, this route represents a transitional pathway between the classic pathway and the more recently acknowledged alternative biosynthesis machinery. The role of the haem biosynthetic pathway in the survival of the bacterium was investigated by testing for inhibitors of HemY. Analogues of acifluorfen are shown to inhibit the flavin-containing HemY, highlighting this protein as a suitable target for the development of drugs against S . aureus. Moreover, the presence of a transitional pathway for haem biosynthesis within many Gram positive pathogenic bacteria suggests that this route has the potential not only for the design of antimicrobials but also for the selective discrimination between bacteria operating different routes to the biosynthesis of haem. [ABSTRACT FROM AUTHOR]

Some bacteria and archaea synthesize haem by an alternative pathway, which involves the sequestration of sirohaem as a metabolic intermediate rather than as a prosthetic group. Along this pathway the two acetic acid side-chains attached to C12 and C18 are decarboxylated by sirohaem decarboxylase, a heterodimeric enzyme composed of AhbA and AhbB, to give didecarboxysirohaem. Further modifications catalysed by two related radical SAM enzymes, AhbC and AhbD, transform didecarboxysirohaem into Fe-coproporphyrin III and haem respectively. The characterization of sirohaem decarboxylase is reported in molecular detail. Recombinant versions of D esulfovibrio desulfuricans, D esulfovibrio vulgaris and M ethanosarcina barkeri AhbA/ B have been produced and their physical properties compared. The D . vulgaris and M . barkeri enzyme complexes both copurify with haem, whose redox state influences the activity of the latter. The kinetic parameters of the D . desulfuricans enzyme have been determined, the enzyme crystallized and its structure has been elucidated. The topology of the enzyme reveals that it shares a structural similarity to the AsnC/ Lrp family of transcription factors. The active site is formed in the cavity between the two subunits and a AhbA/ B-product complex with didecarboxysirohaem has been obtained. A mechanism for the decarboxylation of the kinetically stable carboxyl groups is proposed. [ABSTRACT FROM AUTHOR]

It has recently been shown that the biosynthetic route for both the d1-haem cofactor of dissimilatory cd1 nitrite reductases and haem, via the novel alternative-haem-synthesis pathway, involves siroheme as an intermediate, which was previously thought to occur only as a cofactor in assimilatory sulphite/nitrite reductases. In many denitrifiers (which require d1-haem), the pathway to make siroheme remained to be identified. Here we identify and characterize a sirohydrochlorin-ferrochelatase from P aracoccus pantotrophus that catalyses the last step of siroheme synthesis. It is encoded by a gene annotated as cbiX that was previously assumed to be encoding a cobaltochelatase, acting on sirohydrochlorin. Expressing this chelatase from a plasmid restored the wild-type phenotype of an E scherichia coli mutant-strain lacking sirohydrochlorin-ferrochelatase activity, showing that this chelatase can act in the in vivo siroheme synthesis. A Δ cbiX mutant in P . denitrificans was unable to respire anaerobically on nitrate, proving the role of siroheme as a precursor to another cofactor. We report the 1.9 Å crystal structure of this ferrochelatase. In vivo analysis of single amino acid variants of this chelatase suggests that two histidines, His127 and His187, are essential for siroheme synthesis. This CbiX can generally be identified in α-proteobacteria as the terminal enzyme of siroheme biosynthesis. [ABSTRACT FROM AUTHOR]

Many algae are auxotrophs for vitamin B12 (cobalamin), which they need as a cofactor for B12-dependent methionine synthase (METH). Because only prokaryotes can synthesize the cobalamin, they must be the ultimate source of the vitamin. In the laboratory, a direct interaction between algae and heterotrophic bacteria has been shown, with bacteria supplying cobalamin in exchange for fixed carbon. Here we establish a system to study this interaction at the molecular level. In a culture of a B12-dependent green alga Chlamydomonas nivalis, we found a contaminating bacterium, identified by 16S rRNA analysis as Mesorhizobium sp. Using the sequenced strain of M. loti (MAFF303099), we found that it was able to support the growth of B12-dependent Lobomonas rostrata, another green alga, in return for fixed carbon. The two organisms form a stable equilibrium in terms of population numbers, which is maintained over many generations in semi-continuous culture, indicating a degree of regulation. However, addition of either vitamin B12 or a carbon source for the bacteria perturbs the equilibrium, demonstrating that the symbiosis is mutualistic and facultative. Chlamydomonas reinhardtii does not require B12 for growth because it encodes a B12-independent methionine synthase, METE, the gene for which is suppressed by addition of exogenous B12. Co-culturing C. reinhardtii with M. loti also results in reduction of METE expression, demonstrating that the bacterium can deliver the vitamin to this B12-independent alga. We discuss the implications of this for the widespread distribution of cobalamin auxotrophy in the algal kingdom. [ABSTRACT FROM AUTHOR]

The novel cytochrome P450/redox partner fusion enzyme CYP116B1 from Cupriavidus metallidurans was expressed in and purified from Escherichia coli. Isolated CYP116B1 exhibited a characteristic Fe(II)CO complex with Soret maximum at 449 nm. EPR and resonance Raman analyses indicated low-spin, cysteinate-coordinated ferric haem iron at both 10 K and ambient temperature, respectively, for oxidized CYP116B1. The EPR of reduced CYP116B1 demonstrated stoichiometric binding of a 2Fe-2S cluster in the reductase domain. FMN binding in the reductase domain was confirmed by flavin fluorescence studies. Steady-state reduction of cytochrome c and ferricyanide were supported by both NADPH/NADH, with NADPH used more efficiently ( Km[NADPH] = 0.9 ± 0.5 μ m and Km[NADH] = 399.1 ± 52.1 μ m). Stopped-flow studies of NAD(P)H-dependent electron transfer to the reductase confirmed the preference for NADPH. The reduction potential of the P450 haem iron was -301 ± 7 mV, with retention of haem thiolate ligation in the ferrous enzyme. Redox potentials for the 2Fe-2S and FMN cofactors were more positive than that of the haem iron. Multi-angle laser light scattering demonstrated CYP116B1 to be monomeric. Type I (substrate-like) binding of selected unsaturated fatty acids (myristoleic, palmitoleic and arachidonic acids) was shown, but these substrates were not oxidized by CYP116B1. However, CYP116B1 catalysed hydroxylation (on propyl chains) of the herbicides S-ethyl dipropylthiocarbamate (EPTC) and S-propyl dipropylthiocarbamate (vernolate), and the subsequent N-dealkylation of vernolate. CYP116B1 thus has similar thiocarbamate-oxidizing catalytic properties to Rhodoccocus erythropolis CYP116A1, a P450 involved in the oxidative degradation of EPTC. [ABSTRACT FROM AUTHOR]

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The article discusses the structure of the bacterial microcompartment (BMC) protein PduT, which is a trimeric bacterial microcompartment protein with a 4Fe-4S cluster-binding site. It claims that BMCs are polyhedral cellular inclusions with a protein shell that houses a particular metabolic process. It also cites the propanediol-utilization (Pdu) microcompartment shell that is made from thousands of hexagonal-shaped protein complexes.

The cytochrome cd nitrite reductase from Paracoccus pantotrophus catalyses the one electron reduction of nitrite to nitric oxide using two heme cofactors. The site of nitrite reduction is the d heme, which is synthesized under anaerobic conditions by using nirECFD-LGHJN gene products. In vivo studies with an unmarked deletion strain, Δ nirF, showed that this gene is essential for cd assembly and consequently for denitrification, which was restored when the Δ nirF strain was complemented with wild-type, plasmid-borne, nirF. Removal of a signal sequence and deletion of a conserved N-terminal Gly-rich motif from the NirF coded on a plasmid resulted in loss of in vivo NirF activity. We demonstrate here that the product of the nirF gene is a periplasmic protein and, hence, must be involved in a late stage of the cofactor biosynthesis. In vitro studies with purified NirF established that it could bind d heme. It is concluded that His41 of NirF, which aligns with His200 of the d heme domain of cd, is essential both for this binding and for the production of d heme; replacement of His41 by Ala, Cys, Lys and Met all gave nonfunctional proteins. Potential functions of NirF are discussed. [ABSTRACT FROM AUTHOR]

Of the 30 biosynthetic steps necessary for the production of cobalamin (vitamin B12), eight involve the addition of S-adenosylmethionine-derived methyl groups to the tetrapyrrole framework. These eight methyl additions are catalysed by six canonical methyltransferase domains and one noncanonical methyltransferase domain. Recombinant forms of four methyltransferases from Rhodobacter capsulatus, CobJ, CobM, CobF and CobL, and of the C-terminal noncanonical domain of CobL (CobL-C) have been crystallized, some in more than one crystal form. Most of the crystals diffracted to beyond 2.5 Å resolution and all are suitable for structure determination. Crystals of CobM and CobJ, which are involved in ring contraction, and of CobL, which is involved in two methylations and decarboxylation, are reported for the first time. [ABSTRACT FROM AUTHOR]

Abstract: NirJ is involved in the transformation of precorrin-2 into heme d 1, although its precise role in the process has not been established. The purified protein was found to contain a 4Fe–4S centre, in line with the prediction that it belongs to the radical SAM class of enzymes. This was further confirmed by binding of S-adenosyl-l-methionine (SAM) to dithionite-reduced NirJ, which resulted in a decrease in the signal intensity and in a shift to higher field of the [4Fe–4S]1+ EPR signal. Significantly, though, this approach also led to the appearance of a small but reproducible organic radical signal that was associated with about 2% of the NirJ molecules and was affected by the incorporation of SAM deuterated at the 5′ adenosyl group. [Copyright &y& Elsevier]

Cobalamin (vitamin B12) production in Bacillus megaterium has served as a model system for the systematic evaluation of single and multiple directed molecular and genetic optimization strategies. Plasmid and genome-based overexpression of genes involved in vitamin B12 biosynthesis, including cbiX, sirA, modified hemA, the operons hemAXCDBL and cbiXJCDETLFGAcysGAcbiYbtuR, and the regulatory gene fnr, significantly increased cobalamin production. To reduce flux along the heme branch of the tetrapyrrole pathway, an antisense RNA strategy involving silencing of the hemZ gene encoding coproporphyrinogen III oxidase was successfully employed. Feedback inhibition of the initial enzyme of the tetrapyrrole biosynthesis, HemA, by heme was overcome by stabilized enzyme overproduction. Similarly, the removal of the B12 riboswitch upstream of the cbiXJCDETLFGAcysGAcbiYbtuR operon and the recombinant production of three different vitamin B12 binding proteins (glutamate mutase GlmS, ribonucleotide triphosphate reductase RtpR and methionine synthase MetH) partly abolished B12-dependent feedback inhibition. All these strategies increased cobalamin production in B. megaterium. Finally, combinations of these strategies enhanced the overall intracellular vitamin B12 concentrations but also reduced the volumetric cellular amounts by placing the organism under metabolic stress. [ABSTRACT FROM AUTHOR]

The synthesis of the modified tetrapyrrole known as d1 haem requires several dedicated proteins which are coded for by a set of genes that are often found adjacent to the structural gene, nirS, for cytochrome cd1 nitrite reductase. NirE, the product of the first gene in the nir biogenesis operon, was anticipated to catalyse the conversion of uroporphyrinogen III into precorrin-2; this was confirmed, but it was shown that this enzyme is less sensitive to product inhibition than similar enzymes that function in other biosynthetic pathways. Sequence analysis suggesting that one of these proteins, NirN, is a c-type cytochrome, and has similarity to the part of cytochrome cd1 that binds d1, was validated by recombinant production and characterization of NirN. A NirN– d1 haem complex was demonstrated to release the cofactor to a semi-apo form of cytochrome cd1 from which d1 was extracted, suggesting a role for NirN in the assembly of cytochrome cd1 (NirS). However, inactivation of nirN surprisingly led to only a marginal attenuation of growth of Paracoccus pantotrophus under anaerobic denitrifying conditions. As predicted, NirC is a c-type cytochrome; it was shown in vitro to be an electron donor to the NirN– d1 complex. [ABSTRACT FROM AUTHOR]

Biosynthesis of heme d1, the essential prosthetic group of the dissimilatory nitrite reductase cytochrome cd1, requires the methylation of the tetrapyrrole precursor uroporphyrinogen III at positions C-2 and C-7. We produced Pseudomonas aeruginosa NirE, a putative S-adenosyl-l-methionine (SAM)-dependent uroporphyrinogen III methyltransferase, as a recombinant protein in Escherichia coli and purified it to apparent homogeneity by metal chelate and gel filtration chromatography. Analytical gel filtration of purified NirE indicated that the recombinant protein is a homodimer. NirE was shown to be a SAM-dependent uroporphyrinogen III methyltransferase that catalyzes the conversion of uroporphyrinogen III into precorrin-2 in vivo and in vitro. A specific activity of 316.8 nmol of precorrin-2 h−1·mg−1 of NirE was found for the conversion of uroporphyrinogen III to precorrin-2. At high enzyme concentrations NirE catalyzed an overmethylation of uroporphyrinogen III, resulting in the formation of trimethylpyrrocorphin. Substrate inhibition was observed at uroporphyrinogen III concentrations above 17 μm. The protein did bind SAM, although not with the same avidity as reported for other SAM-dependent uroporphyrinogen III methyltransferases involved in siroheme and cobalamin biosynthesis. A P. aeruginosa nirE transposon mutant was not complemented by native cobA encoding the SAM-dependent uroporphyrinogen III methyltransferase involved in cobalamin formation. However, bacterial growth of the nirE mutant was observed when cobA was constitutively expressed by a complementing plasmid, underscoring the special requirement of NirE for heme d1 biosynthesis. [ABSTRACT FROM AUTHOR]

The article presents a study on the extinction debt in a declining species by using the metapopulations of the marsh fritillary butterfly Euphydryas aurinia in Great Britain as a model system. It examines whether the decline in the distribution of animal species is keeping pace with landscape change or whether current distributions overestimate the probabilities of future persistence. Results indicate a widespread extinction debt and suggest that metapopulation modeling is a potential means to identify landscapes that are near extinction thresholds for threatened species. Conservation management and reserve designation are recommended for extant metapopulations of a declining species.

Abstract: In the model P450 BM3 system, the P450 is fused to its diflavin reductase partner in a single polypeptide. BM3 dimerizes in solution, but the catalytic relevance of the phenomenon was hitherto unknown. We show that BM3 fatty acid hydroxylase specific activity decreases sharply at low enzyme concentrations, consistent with separation of active dimer into inactive monomer. Reductase-dependent specific activities are maintained or enhanced at low concentration, suggesting inter-flavin electron transfer is unaffected. Fatty acid oxidation is reconstituted by mixing inactive oxygenase (A264H) and FMN-depleted (G570D) mutants, demonstrating that inter-monomer (FMN1-to-heme2) electron transfer supports oxygenase activity in the BM3 dimer. [Copyright &y& Elsevier]

Sirohaem is a tetrapyrrole-derived prosthetic group that is required for the essential assimilation of sulfur and nitrogen into all living systems as part of the sulfite and nitrite reductase systems. The final two steps in the biosynthesis of sirohaem involve a β-NAD+-dependent dehydrogenation of precorrin-2 to generate sirohydrochlorin followed by ferrochelation to yield sirohaem. In Saccharomyces cerevisiae, Met8p is a bifunctional enzyme that carries out both of these reactions. Here, we report the 2.2 Å resolution crystal structure of Met8p, which adopts a novel fold that bears no resemblance to the previously determined structures of cobalt- or ferro-chelatases. Analysis of mutant proteins suggests that both catalytic activities share a single active site, and that Asp141 plays an essential role in both dehydrogenase and chelatase processes. [ABSTRACT FROM AUTHOR]

Describes the construction of heterologous hemA and hemT gene over-expression systems in the photosynthetic bacterium, Rhodobacter sphaeroides. Refolding and purification of hemT; Purification of hemA; Determination of the physical and kinetic properties of hemA; Effect of protein modifying agents on the activity of 5-aminolaevulinic acid synthase.

The Bacillus megaterium cbiF, encoding the cobalt-precorrin-4 S-adenosyl-L-methionine-dependent transmethylase of the anaerobic cobalamin biosynthetic pathway, has been cloned and overexpressed as a His-tagged recombinant protein in Escherichia coli. The protein was purified to homogeneity by a combination of metal chelate chromatography and high-resolution anion-exchange chromatography. The protein migrated with a subunit mass of 31 kDa by SDS/PAGE and with a molecular mass of 62 kDa by analytical gel filtration, suggesting that the native recombinant protein is a homodimer. The His-tagged protein was physiologically active as it was able to complement a Salmonella typhimurium cbiF mutant. However, the protein did not bind S-adenosyl-L-methionine with the same avidity as observed with other corrin biosynthetic transmethylases. A crystallisation screen of the purified protein led to the identification of two discrete crystal forms. One of these forms has been characterised and a full data set collected. [ABSTRACT FROM AUTHOR]

CobE, a protein implicated in vitamin B12 biosynthesis, from Pseudomonas aeruginosa has been overexpressed in Escherichia coil, purified and crystallized using hanging-drop vapour diffusion. The crystals belong to the primitive orthorhombic space group P212121, with unit-cell parameters a = 31.86, b= 41.07, c = 87.41 Å. The diffraction extends to a resolution of 1.9 Å. There is one molecule per asymmetric unit and the estimated solvent content is 35%. SeMet- labelled CobE has been prepared and crystallizes under the same conditions as the native protein with diffraction to 1.7 Å. The anomalous measurements will be used for phasing. [ABSTRACT FROM AUTHOR]

Sirohaem, the prosthetic group of assimilatory sulfite and nitrite reductases, is a modified tetrapyrrole that belongs to the same fraternity of metallo-prosthetic groups as haem, chlorophyll, cobalamin and coenzyme F430 [Warren & Scott (1990), Trends Biochem Sci. 15, 486-491]. In Saccharomyces cerevisiae, the last step in the biosynthesis of sirohaem involves Met8p, a bifunctional enzyme responsible for both the NAD+-dependent dehydrogenation of the corrin ring and ferrochelation. Optimization of the protein storage buffer according to the results of crystallization trials resulted in a more monodisperse protein solution. Crystals were grown that diffracted to 2.1 Å. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented in response to the article "Europe needs a new vision for a Natura 2020 network" by A. Hochkirch and colleagues that was published in a previous 2013 issue, which discusses laws concerning the conservation of birds and habitats.

B12 ist ein einzigartiges Vitamin, da es nur von bestimmten Prokaryoten synthetisiert werden kann. Die Komplexität seiner Biosynthese resultiert aus der für biochemische Prozesse essenziellen Cobalt ‐ Corrinstruktur, die dem Coenzym B12 (AdoCbl) die Fähigkeit zur Katalyse enzymatischer Radikalreaktionen verleiht. Was macht aber gerade Cobalt so passend für seine Rolle in B12 ‐ abhängigen Enzymen? Um dieser Frage nachzugehen, wurde Cobalt in AdoCbl gegen Rhodium ausgetauscht; hierfür wurde das Rh ‐ Analog 5′ ‐ Desoxy ‐ 5′ ‐ adenosylrhodibalamin (AdoRbl) de novo mittels kombinierter biologischer und chemischer Totalsynthese hergestellt. AdoRbl zeigte keine Aktivität in einem mikrobiellen Methionin ‐ Synthase ‐ Bioassay in vivo und war ein In ‐ vitro ‐ Inhibitor einer AdoCbl ‐ abhängigen Diol ‐ Dehydratase. NMR ‐ Analysen zeigten hohe Strukturähnlichkeit von AdoRbl mit AdoCbl. Beim Vergleich der Strukturen im Kristall wurde aber festgestellt, dass RhIII besser in den Corrinring passt als CoIII, was Fragen zur gegenwärtigen Sicht über die Evolution der Corrine aufwirft. Rhodium & Co.: Das Rhodiumhomologe (AdoRbl) von Coenzym B12 (AdoCbl) wurde totalsynthetisch mittels einer Kombination biosynthetischer und chemisch ‐ synthetischer Schritte hergestellt. AdoRbl war inaktiv als Cofaktor. Kristallstrukturen von AdoRbl und AdoCbl zufolge passt RhIII besser in den Corrinliganden als CoIII. Dies wirft Fragen zur angenommenen Strukturanpassung des Corrins an das „Kobold” ‐ Element Cobalt auf. [ABSTRACT FROM AUTHOR]

B12 is unique among the vitamins as it is biosynthesized only by certain prokaryotes. The complexity of its synthesis relates to its distinctive cobalt corrin structure, which is essential for B12 biochemistry and renders coenzyme B12 (AdoCbl) so intriguingly suitable for enzymatic radical reactions. However, why is cobalt so fit for its role in B12-dependent enzymes? To address this question, we considered the substitution of cobalt in AdoCbl with rhodium to generate the rhodium analogue 5′-deoxy-5′-adenosylrhodibalamin (AdoRbl). AdoRbl was prepared by de novo total synthesis involving both biological and chemical steps. AdoRbl was found to be inactive in vivo in microbial bioassays for methionine synthase and acted as an in vitro inhibitor of an AdoCbl-dependent diol dehydratase. Solution NMR studies of AdoRbl revealed a structure similar to that of AdoCbl. However, the crystal structure of AdoRbl revealed a conspicuously better fit of the corrin ligand for RhIII than for CoIII, challenging the current views concerning the evolution of corrins. [ABSTRACT FROM AUTHOR]