Novel lipoate-binding proteins and their role in sulfur oxidation
Creators: name: Tanabe, Tomohisa Sebastian
Publisher: name: Universitäts- und Landesbibliothek Bonn
Year: 2024
Subjects: subject: Lipoate
subject: Hyphomicrobium denitrificans
subject: sulfur
subject: sulfur oxidation
subject: sHdr
subject: heterodisulfide reduktase
subject: HMSSS
subject: HMSS2
subject: ddc:570

description: The global sulfur cycle is a highly complex network of interconnected processes that involve the conversion of organic and inorganic sulfur compounds across multiple redox states. Driven by prokaryotes through the oxidation, reduction, or even disproportionation of organic and inorganic sulfur compounds it plays a crucial role in maintaining a balanced biosphere. Exploring these processes and the involved prokaryotes is highly relevant for the understanding of current and ancient ecosystems. In recent years, the development of next-generation sequencing technologies and genome reconstruction from metagenomes has led to an increasing number of accessible prokaryotic genomes from a previously unknown diversity of species. Assigning metabolic pathways to this genetic information based on experimentally validated functions has become one of the most important tasks to make use of the increasing amount of data. This work combines bioinformatic and biochemical techniques to explore the dissimilatory sulfur oxidation. <br> For the prediction genes and gene clusters with a function in dissimilatory sulfur oxidation, or reduction in (meta-)genomes a software named HMS-S-S was developed. With the advanced version HMSS2 proteins involved in the conversion, degradation and transport of organic sulfur compounds were added. Both tools were shown to predict genes and gene clusters with a reasonable confidence using profiled hidden Markov models. The generation of profiled hidden Markov models was also automated as an independent software to keep up with the discovery of new metabolic pathways. <br> These tools were used to elucidate the mechanisms of a dissimilatory sulfur oxidation through a sulfur-oxidizing heterodisulfide reductase (sHdr) complex that interacts with a lipoate-binding protein (LbpA). Before it is functionally active, the lipoate cofactor must be covalently attached to its cognate enzyme. Here, a novel LbpA-specific lipoate biosynthesis pathway was demonstrated to be active in bacteria using a genetic approach. This pathway operated autonomously and concurrently with the canonical lipoate biosynthesis pathways. Examining the ocurrence of the novel and canonical lipoate biosynthesis across prokaryotic biodiversity revealed a much broader distribution of the lipoate assembly systems than previously recognized and located the evolutionary origin of the novel biosynthesis pathway inside the archaeal domain. Genetically the sHdr system is tightly connected to several sulfur transferases, which are proposed to relay protein-bound sulfur to the LbpA-sHdr complex. Indeed it was possible to detect sulfane sulfur transfer activity and to determine the catalytically active cysteines. With sulfur transfer assays it was possible to reconstruct the sulfur relay system of four bacteria. Assessing the co-occurrence of these sulfur transferases in the <em>shdr</em> gene cluster revealed a general importance of these sulfur relay systems. Two regulatory sulfur-sensing proteins were also characterized, which likely receive sulfur from these sulfur transferases. Additionally, the co-occurrence of the sHdr system and other sulfur-oxidizing systems was examined. In total this combination of genetic, biochemical and bioinformatic methods contributed to the understanding of the complexity of microbial sulfur-oxidation.