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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.