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The NadABC pathway is involved in the biosynthesis of nicotinamide adenine dinucleotide (NAD) and is a dominant pathway in bacteria. The conversion of l-aspartate to quinolinic acid is initiated by the l-aspartate oxidase NadB, which catalyzes the formation of iminoaspartate that is used by quinolinate synthase NadA in a condensation reaction with dihydroxyacetone phosphate to produce quinolinic acid. NadA is a [4Fesingle bond4S] cluster-containing enzyme that is indispensable in the production of NAD. In B. subtilis, the cysteine sulfurtransferase nifS gene is located in genomic proximity to the nad genes, and its expression is regulated by NadR based on the availability of nicotinic acid. Inactivation of nifS leads to inactivation of the NAD pathway and, consequently, nicotinic acid auxotrophy. In this study, we explored the hypothesis that NifS’ involvement in NAD biosynthesis is associated with its role in the maturation of NadA [4Fesingle bond4S] cluster. We showed through in vitro reconstitution experiments that NifS is catalytically competent in promoting cluster assembly onto apo-NadA and that the rate of reactivation depends on the rate of sulfur mobilization. Furthermore, the activity of NifS in sulfur mobilization is modulated by Apo-NadA. Under conditions of cluster synthesis, apo-NadA enhances the turnover rate of NifS. This phenomenon is not observed for YrvO, NifZ, and SufSU, the other three cysteine sulfurtransferases in B. subtilis. This work provides biochemical evidence for the requirement of a dedicated cysteine desulfurase in the maturation of specialized Fesingle bondS enzymes. 

Low-molecular-weight (LMW) thiols play critical roles in maintaining redox buffer systems required for normal biological function. Glutathione (GSH) represents the most common LMW thiol found in Nature, but Gram-positive bacteria utilize bacillithiol (BSH) or mycothiol (MSH). Nitroxyl (HNO) can influence bacterial transcription through persulfide formation, a biological phenomenon that prompts the examination of the reactions of HNO with these LMW thiols. The development and application of colorimetric and enzymatic (Bacillus subtilis thioredoxin assay) methods combined with mass spectrometry of reaction products show the unique reactivity of BSH to favor sulfinamide adduct formation upon equimolar reaction with HNO. The reaction profile with GSH results in nearly equal distribution between sulfinamide:disulfide, whereas reaction with MSH only yields disulfide. These varied results led to the preparation of a group of BSH and MSH analogs, and their reactions with HNO reveal the requirement for a free amine group for sulfinamide formation. The thiol and amine group pKa's appear critical for sulfinamide generation, with the thiolate acting as a nucleophile to attack HNO and the ammonium donating a proton to facilitate water loss from the N-hydroxysulfenamide intermediate. Furthermore, the B. subtilis thioredoxin system efficiently reduces BSSB with a calculated KM_BSSB = 34 ± 3 μM and Vmax = 152 ± 3.4 nmol/min/nmol TrxR (kcat = 2.5 s−1), but does not reduce bacillithiol sulfinamide. Similarly, this thioredoxin reduces MSSM with a calculated KM_MSSM of 9 ± 2.1 μM and Vmax of 103 ± 7.1 nmol/min/nmol TrxR (kcat = 1.7 s−1). Bacillithiol possesses a unique structure that allows a rapid reaction with HNO to form a stable product that may provide a basis for antibiotic development or clues for further biological roles of nitroxyl. 

The biological synthesis of iron–sulfur (Fe–S) clusters requires dedicated pathways involved in the recruitment and activation of Fe and S for cluster assembly with subsequent transfer of preformed clusters to acceptor proteins. Several pathways have been described that include various numbers and types of biosynthetic components, although all of them share the same basic principles for [Fe–S] cluster formation and delivery to target proteins. The NifUS system was discovered and first described in studies involving the model diazotroph Azotobacter vinelandii . It has a dedicated role in serving as the starting point for the activation of [Fe–S] cluster-containing proteins specifically involved in biological nitrogen fixation. NifS is a pyridoxal-5′-phosphate containing l -cysteine-dependent sulfur transferase that delivers activated sulfur to the three-domain NifU, which not only serves as a scaffold for the construction of [2Fe–2S] and [4Fe–4S] clusters but also participates in their delivery to various target proteins involved in nitrogen fixation. Interestingly, analysis of sequenced genomes reveals that the three-domain NifU and NifU-like encoded proteins are not limited to diazotrophs, suggesting a broader role for this system in [Fe–S] cluster biogenesis in other organisms. The colocalization of adjacent nifU and nifS encoding sequences in most of these genomes also provides a strong indication for the involvement of the NifU–NifS [Fe–S] cluster assembly and delivery toolkit for activation of [Fe–S] cluster-containing proteins in a variety of organisms that do not fix nitrogen. 

Sulfur-containing biomolecules such as [Fe-S] clusters, thiamin, biotin, molybdenum cofactor, and sulfur-containing tRNA nucleosides are essential for various biochemical reactions. The amino acid l-cysteine serves as the major sulfur source for the biosynthetic pathways of these sulfur-containing cofactors in prokaryotic and eukaryotic systems. The first reaction in the sulfur mobilization involves a class of pyridoxal-5′-phosphate (PLP) dependent enzymes catalyzing a Cys:sulfur acceptor sulfurtransferase reaction. The first half of the catalytic reaction involves a PLP-dependent single bondS bond cleavage, resulting in a persulfide enzyme intermediate. The second half of the reaction involves the subsequent transfer of the thiol group to a specific acceptor molecule, which is responsible for the physiological role of the enzyme. Structural and biochemical analysis of these Cys sulfurtransferase enzymes shows that specific protein-protein interactions with sulfur acceptors modulate their catalytic reactivity and restrict their biochemical functions. 

Bacillithiol (BSH) replaces glutathione (GSH) as the most prominent low-molecular-weight thiol in many low G + C gram-positive bacteria. BSH plays roles in metal binding, protein/ enzyme regulation, detoxification, redox buffering, and bacterial virulence. Given the small amounts of BSH isolated from natural sources and relatively lengthy chemical syntheses, the reactions of BSH with pertinent reactive oxygen, nitrogen, and sulfur species remain largely unexplored. We prepared BSH and exposed it to nitroxyl (HNO), a reactive nitrogen species that influences bacterial sulfur metabolism. The profile of this reaction was distinct from HNO oxidation of GSH, which yielded mixtures of disulfide and sulfinamide. The reaction of BSH and HNO (generated from Angeli’s salt) gives only sulfinamide products, including a newly proposed cyclic sulfinamide. Treatment of a glucosamine−cysteine conjugate, which lacks the malic acid group, with HNO forms disulfide, implicating the malic acid group in sulfinamide formation. This finding supports a mechanism involving the formation of an N-hydroxysulfenamide intermediate that dehydrates to a sulfenium ion that can be trapped by water or internally trapped by an amide nitrogen to give the cyclic sulfinamide. The biological relevance of BSH reactivity toward HNO is provided through in vivo experiments demonstrating that Bacillus subtilis exposed to HNO shows a growth phenotype, and a strain unable to produce BSH shows hypersensitivity toward HNO in minimal medium cultures. Thiol analysis of HNO-exposed cultures shows an overall decrease in reduced BSH levels, which is not accompanied by increased levels of BSSB, supporting a model involving the formation of an oxidized sulfinamide derivative, identified in vivo by high-pressure liquid chromatography/mass spectrometry. Collectively, these findings reveal the unique chemistry and biology of HNO with BSH in bacteria that produce this biothiol. 

The wobble bases of tRNAs that decode split codons are often heavily modified. In bacteria, tRNA^{Glu, Gln, Asp}contains a variety of xnm⁵s²U derivatives. The synthesis pathway for these modifications is complex and fully elucidated only in a handful of organisms, including the Gram-negativeEscherichia coliK12 model. Despite the ubiquitous presence of mnm⁵s²U modification, genomic analysis shows the absence ofmnmCorthologous genes, suggesting the occurrence of alternate biosynthetic schemes for the conversion of cmnm⁵s²U to mnm⁵s²U. Using a combination of comparative genomics and genetic studies, a member of the YtqA subgroup of the radical Sam superfamily was found to be involved in the synthesis of mnm⁵s²U in bothBacillus subtilisandStreptococcus mutans. This protein, renamed MnmL, is encoded in an operon with the recently discovered MnmM methylase involved in the methylation of the pathway intermediate nm⁵s²U into mnm⁵s²U inB. subtilis. Analysis of tRNA modifications of bothS. mutansandStreptococcus pneumoniaeshows that growth conditions and genetic backgrounds influence the ratios of pathway intermediates owing to regulatory loops that are not yet understood. The MnmLM pathway is widespread along the bacterial tree, with some phyla, such as Bacilli, relying exclusively on these two enzymes. Although mechanistic details of these newly discovered components are not fully resolved, the occurrence of fusion proteins, alternate arrangements of biosynthetic components, and loss of biosynthetic branches provide examples of biosynthetic diversity to retain a conserved tRNA modification in Nature.IMPORTANCEThe xnm⁵s²U modifications found in several tRNAs at the wobble base position are widespread in bacteria where they have an important role in decoding efficiency and accuracy. This work identifies a novel enzyme (MnmL) that is a member of a subgroup of the very versatile radical SAM superfamily and is involved in the synthesis of mnm⁵s²U in several Gram-positive bacteria, including human pathogens. This is another novel example of a non-orthologous displacement in the field of tRNA modification synthesis, showing how different solutions evolve to retain U34 tRNA modifications.