CobT and BzaC catalyze the regiospecific activation and methylation of the 5-hydroxybenzimidazole lower ligand in anaerobic cobamide biosynthesis

Vitamin B12 and other cobamides are essential cofactors required by many organisms and are synthesized by a subset of prokaryotes via distinct aerobic and anaerobic routes. The anaerobic biosynthesis of 5,6-dimethylbenzimidazole (DMB), the lower ligand of vitamin B12, involves five reactions catalyzed by the bza operon gene products, namely the hydroxybenzimidazole synthase BzaAB/BzaF, phosphoribosyltransferase CobT, and three methyltransferases, BzaC, BzaD, and BzaE, that conduct three distinct methylation steps. Of these, the methyltransferases that contribute to benzimidazole lower ligand diversity in cobamides remain to be characterized, and the precise role of the bza operon protein CobT is unclear. In this study, we used the bza operon from the anaerobic bacterium Moorella thermoacetica (comprising bzaA-bzaB-cobT-bzaC) to examine the role of CobT and investigate the activity of the first methyltransferase, BzaC. We studied the phosphoribosylation catalyzed by MtCobT and found that it regiospecifically activates 5-hydroxybenzimidazole (5-OHBza) to form the 5-OHBza-ribotide (5-OHBza-RP) isomer as the sole product. Next, we characterized the domains of MtBzaC and reconstituted its methyltransferase activity with the predicted substrate 5-OHBza and with two alternative substrates, the MtCobT product 5-OHBza-RP and its riboside derivative 5-OHBza-R. Unexpectedly, we found that 5-OHBza-R is the most favored MtBzaC substrate. Our results collectively explain the long-standing observation that the attachment of the lower ligand in anaerobic cobamide biosynthesis is regiospecific. In conclusion, we validate MtBzaC as a SAM:hydroxybenzimidazole-riboside methyltransferase (HBIR-OMT). Finally, we propose a new pathway for the synthesis and activation of the benzimidazolyl lower ligand in anaerobic cobamide biosynthesis.

Anion Inhibition Studies of the Beta-Carbonic Anhydrase from Escherichia coli

The interconversion of CO2 and HCO3− is catalyzed by a superfamily of metalloenzymes, known as carbonic anhydrases (CAs, EC 4.2.1.1), which maintain the equilibrium between dissolved inorganic CO2 and HCO3−. In the genome of Escherichia coli, a Gram-negative bacterium typically colonizing the lower intestine of warm-blooded organisms, the cyn operon gene includes the CynT gene, encoding for a β-CA, and CynS gene, encoding for the cyanase. CynT (β-CA) prevents the depletion of the cellular bicarbonate, which is further used in the reaction catalyzed by cyanase. A second β-CA (CynT2 or Can or yadF), as well as a γ and ι-CAs were also identified in the E. coli genome. CynT2 is essential for bacterial growth at atmospheric CO2 concentration. Here, we characterized the kinetic properties and the anion inhibition profiles of recombinant CynT2. The enzyme showed a good activity for the physiological CO2 hydratase reaction with the following parameters: kcat = 5.3 × 105 s−1 and kcat/KM = of 4.1 × 107 M−1 s−1. Sulfamide, sulfamate, phenylboronic acid, phenylarsonic acid, and diethyldithiocarbamate were the most effective CynT2 inhibitors (KI = 2.5 to 84 µM). The anions allowed for a detailed understanding of the interaction of inhibitors with the amino acid residues surrounding the catalytic pocket of the enzyme and may be used as leads for the design of more efficient and specific inhibitors.

Deep evolutionary origin of nematode SL2 trans-splicing revealed by genome-wide analysis of the Trichinella spiralis transcriptome

ABSTRACTSpliced leader trans-splicing is intimately associated with the presence of eukaryotic operons, allowing the processing of polycistronic RNAs into individual mRNAs. Most of our understanding of spliced leader trans-splicing as it relates to operon gene expression comes from studies in C. elegans. In this organism, two distinct spliced leader trans-splicing events are recognised: SL1, which is used to replace the 5’ ends of pre-mRNAs that have a nascent monomethyl guanosine cap; and SL2, which provides the 5’ end to uncapped pre-mRNAs derived from polycistronic RNAs. Limited data on operons and spliced leader trans-splicing in other nematodes suggested that SL2-type trans-splicing is a relatively recent innovation, associated with increased efficiency of polycistronic processing, and confined to only one of the five major nematode clades, Clade V. We have conducted the first transcriptome-wide analysis of spliced leader trans-splicing in a nematode species, Trichinella spiralis, which belongs to a clade distantly related to Clade V. Our work identifies a set of T. spiralis SL2-type spliced leaders that are specifically used to process polycistronic RNAs, the first examples of specialised spliced leaders that have been found outside of Clade V. These T. spiralis spliced leader RNAs possess a perfectly conserved stem-loop motif previously shown to be essential for polycistronic RNA processing in C. elegans. We show that this motif is found in specific sets of spliced leader RNAs broadly distributed across the nematode phylum. This work substantially revises our understanding of the evolution of nematode spliced leader trans-splicing, showing that the machinery for SL2 trans-splicing evolved much earlier during nematode evolution than was previously appreciated, and has been conserved throughout the radiation of the nematode phylum.

The Mannitol Utilization System of the Marine Bacterium Zobellia galactanivorans

ABSTRACTMannitol is a polyol that occurs in a wide range of living organisms, where it fulfills different physiological roles. In particular, mannitol can account for as much as 20 to 30% of the dry weight of brown algae and is likely to be an important source of carbon for marine heterotrophic bacteria.Zobellia galactanivorans(Flavobacteriia) is a model for the study of pathways involved in the degradation of seaweed carbohydrates. Annotation of its genome revealed the presence of genes potentially involved in mannitol catabolism, and we describe here the biochemical characterization of a recombinant mannitol-2-dehydrogenase (M2DH) and a fructokinase (FK). Among the observations, the M2DH ofZ. galactanivoranswas active as a monomer, did not require metal ions for catalysis, and featured a narrow substrate specificity. The FK characterized was active on fructose and mannose in the presence of a monocation, preferentially K+. Furthermore, the genes coding for these two proteins were adjacent in the genome and were located directly downstream of three loci likely to encode an ATP binding cassette (ABC) transporter complex, suggesting organization into an operon. Gene expression analysis supported this hypothesis and showed the induction of these five genes after culture ofZ. galactanivoransin the presence of mannitol as the sole source of carbon. This operon for mannitol catabolism was identified in only 6 genomes ofFlavobacteriaceaeamong the 76 publicly available at the time of the analysis. It is not conserved in allBacteroidetes; some species contain a predicted mannitol permease instead of a putative ABC transporter complex upstream of M2DH and FK ortholog genes.