Vitamin B 12 (cobalamin) is required by humans and other organisms for diverse metabolic processes, although only a subset of prokaryotes is capable of synthesizing B 12 and other cobamide cofactors. The complete aerobic and anaerobic pathways for the de novo biosynthesis of B 12 are known, with the exception of the steps leading to the anaerobic biosynthesis of the lower ligand, 5,6-dimethylbenzimidazole (DMB). Here, we report the identification and characterization of the complete pathway for anaerobic DMB biosynthesis. This pathway, identified in the obligate anaerobic bacterium Eubacterium limosum, is composed of five previously uncharacterized genes, bzaABCDE, that together direct DMB production when expressed in anaerobically cultured Escherichia coli. Expression of different combinations of the bza genes revealed that 5-hydroxybenzimidazole, 5-methoxybenzimidazole, and 5-methoxy-6-methylbenzimidazole, all of which are lower ligands of cobamides produced by other organisms, are intermediates in the pathway. The bza gene content of several bacterial and archaeal genomes is consistent with experimentally determined structures of the benzimidazoles produced by these organisms, indicating that these genes can be used to predict cobamide structure. The identification of the bza genes thus represents the last remaining unknown component of the biosynthetic pathway for not only B 12 itself, but also for three other cobamide lower ligands whose biosynthesis was previously unknown. Given the importance of cobamides in environmental, industrial, and human-associated microbial metabolism, the ability to predict cobamide structure may lead to an improved ability to understand and manipulate microbial metabolism.

MoaA, a radical S-adenosylmethionine (SAM) enzyme, catalyzes the first step in molybdopterin biosynthesis. This reaction involves a complex rearrangement in which C8 of guanosine triphosphtate is inserted between the C2′ and the C3′ carbons of the ribose. This study identifies the site of initial hydrogen atom abstraction by the adenosyl radical and advances a mechanistic proposal for this unprecedented reaction.

In this minireview, we describe the radical S-adenosylmethionine enzymes involved in the biosynthesis of thiamin, menaquinone, molybdopterin, coenzyme F 420 , and heme. Our focus is on the remarkably complex organic rearrangements involved, many of which have no precedent in organic or biological chemistry. Radical S-adenosylmethionine (SAM)2 enzymes are among the most intriguing enzymes discovered over the past 15 years. In these enzymes, reduction of SAM by a 1ϩ cluster generates the adenosyl radical, which then abstracts a hydrogen atom from the substrate. The resulting substrate radical usually undergoes a rearrangement or fragmentation reaction to give the product radical (1). Sequence analysis suggests that a large number of enzymes use the radical SAM catalytic motif (2). We are still at an early stage of defining the catalytic mechanisms and the structural enzymology of this fascinating enzyme family.This minireview focuses on the organic chemistry of the radical SAM enzymes involved in cofactor biosynthesis: thiamin (1), F 0 (2), menaquinone (3), molybdopterin (4), and heme (5) (Fig. 1). (Biotin and lipoic biosynthesis also uses radical SAM enzymology and is covered separately in another minireview in this series (42).) In contrast to iron(IV)-oxo-derived radicals, where the dominant chemistry involves radical recombination with the iron-bound oxygen (P 450 rebound rate of Ͼ10 10 s Ϫ1 ) (3), radicals formed by hydrogen atom transfer to the 5Ј-deoxyadenosyl (5Ј-dA) radical are more persistent because the reverse reaction is relatively slow. This allows time for complex rearrangements to occur. Radical SAM enzymes catalyze a remarkable range of reactions due to the high intrinsic reactivity of organic radicals, which can undergo rapid hydrogen atom abstraction, double-bond addition, and fragmentation reactions as shown in Fig. 2. Prokaryotic Thiamin Pyrimidine Synthase (ThiC)Thiamin pyrophosphate (1) is an important cofactor in carbohydrate metabolism and branched chain amino acid biosynthesis, where it plays a key role in stabilization of the acyl carbanion biosynthon. The thiamin pyrimidine synthase (ThiC) catalyzes the conversion of aminoimidazole ribotide (AIR; 6) to hydroxymethylpyrimidine phosphate (HMP-P; 8) (Fig. 3A) (4, 5). The origin of all of the atoms of the product and the fate of all of the atoms of the substrate have been determined using isotope labeling studies (6 -8). This rearrangement, as far as we can tell, is the most complex unsolved rearrangement in primary metabolism.A mechanistic proposal for this reaction is shown in Fig. 3B (5). In this proposal, radical 7 abstracts a hydrogen atom from 6 to form 10. A ␤-scission followed by N-glycosyl bond cleavage gives 12. Electrophilic addition to the aminoimidazole followed by hydrogen atom transfer gives 14. The regenerated 5Ј-dA radical (7) abstracts a hydrogen atom from 14 to form 15. Radical addition to the imine followed by ␤-scission gives 17. A second ␤-scission followed by a diol dehydratase-like rearrangement gives 20 and 21. Ra...

It has been hypothesized that mitochondria evolved from a bacterial ancestor that initially became established in a protoeukaryotic cell as an endosymbiont. Here we model this first stage of mitochondrial evolution by engineering endosymbiosis between E. coli and the budding yeast S. cerevisiae. Fusion of yeast with E. coli ectopically expressing several genes from unrelated, intracellular bacteria was key for establishing endosymbiosis. ADP/ATP translocase‐expressing E. coli provided an energy source for a respiration‐deficient cox2 yeast mutant, enabling growth of yeast‐E. coli chimera on a non‐fermentable carbon source. Similarly, yeast provided a source of thiamin or NAD to an E. coli thiamin or NAD auxotroph respectively. Expression of several SNARE‐like protein on the surface of E. coli was also required to prevent lysosomal degradation. The engineered yeast‐E. coli chimeras sustained growth on selection medium in containing the antibiotic carbenicillin indicating the presence of intracellular E. coli which supports the growth by ATP synthesis on selection medium. Further, sf‐gfp expressing E. coli endosymbionts could be observed in the yeast by super resolution fluorescence microscopy after more than 40 doublings. This readily manipulated system should allow us to experimentally delineate host‐endosymbiont adaptations that occurred during evolution of the current much reduced mitochondrial genome This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Nisin is a complex lanthipeptide that has broad spectrum antibacterial activity. In efforts to broaden the structural diversity of this ribosomally synthesized lantibiotic, we now report the recombinant expression of Nisin variants that incorporate non-canonical amino acids (ncAAs) at discrete positions. This is achieved by expressing the nisA structural gene, cyclase (nisC) and dehydratase (nisB), together with an orthogonal nonsense suppressor tRNA/aminoacyl-tRNA synthetase pair in E.coli. A number of ncAAs with novel chemical reactivity were genetically incorporated into NisA, including an α-chloroacetamide-containing ncAA which allowed for the expression of Nisin variants with novel macrocyclic topologies. This methodology should allow for the exploration of lanthipeptide variants with new or enhanced activities.

The ability to add noncanonical amino acids to the genetic code may allow one to evolve proteins with new or enhanced properties using a larger set of building blocks. To this end, we have been able to select mutant proteins with enhanced thermal properties from a library of E. coli homoserine o-succinyltransferase (metA) mutants containing randomly incorporated noncanonical amino acids. Here, we show that substitution of Phe 21 with p-benzoylphenylalanine (pBzF), increases the melting temperature of E. coli metA by 21°C. This dramatic increase in thermal stability, arising from a single mutation, likely results from a covalent adduct between Cys 90 and the keto group of pBzF that stabilizes the dimeric form of the enzyme. These experiments show that an expanded genetic code can provide unique solutions to the evolution of proteins with enhanced properties.

Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to generate a 5′-deoxyadenosyl radical. Canonical radical SAM enzymes are characterized by a β-barrel-like fold and SAM anchors to the differentiated iron of the cluster, which is located near the amino terminus and within the β-barrel, through its amino and carboxylate groups. Here we show that ThiC, the thiamin pyrimidine synthase in plants and bacteria, contains a tethered cluster-binding domain at its carboxy terminus that moves in and out of the active site during catalysis. In contrast to canonical radical SAM enzymes, we predict that SAM anchors to an additional active site metal through its amino and carboxylate groups. Superimposition of the catalytic domains of ThiC and glutamate mutase shows that these two enzymes share similar active site architectures, thus providing strong evidence for an evolutionary link between the radical SAM and adenosylcobalamin-dependent enzyme superfamilies.

Prokaryotic and eukaryotic genomic DNA is comprised of the four building blocks A, G, C and T. We have begun to explore the consequences of replacing a large fraction or all of a nucleoside in genomic DNA with a modified nucleoside. As a first step we have investigated the possibility of replacement of T by 2′-deoxy-5-(hydroxymethyl)uridine (5hmU) in the genomic DNA of E. coli. Metabolic engineering with phage genes followed by random mutagenesis enabled us to achieve approximately 75% replacement of T by 5hmU in the E. coli genome and in plasmids.