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type:Review, Sulfur is an essential element required for plant growth. It can be found as a thiol group of proteins or non-protein molecules, and as various sulfur-containing small biomolecules, including iron-sulfur (Fe/S) clusters, molybdenum cofactor (Moco), and sulfur-modified nucleotides. Thiol-mediated redox regulation has been well investigated, whereas biosynthesis pathways of the sulfur-containing small biomolecules have not yet been clearly described. In order to understand overall sulfur transfer processes in plant cells, it is important to elucidate the relationships among various sulfur delivery pathways as well as to investigate their interactions. In this review, we summarize the information from recent studies on the biosynthesis pathways of several sulfur-containing small biomolecules and the proteins participating in these processes. In addition, we show characteristic features of gene expression in Arabidopsis at the early stage of sulfate depletion from the medium, and we provide insights into sulfur transfer processes in plant cells.

For many decades, it was thought that the phenomenon of cellular compartmentalization was exclusive to eukaryotes. Advances in cell imaging techniques have revealed that the presence of subcellular compartments is widespread throughout prokaryotes and that there exists a great diversity of organelles in bacteria and archaea. Recently, a new class of prokaryotic organelle has been discovered – the protein-bounded prokaryotic nanocompartments, also called encapsulins. Encapsulins are among the simplest of the prokaryotic organelles, often comprised of a two gene system; a shell-encoding gene and a cargo-encoding gene. Despite their simplicity, little is known about the biochemistry and physiological function of the encapsulins.Here we investigate the structure and function of the encapsulin nanocompartments from various prokaryotes. First, we demonstrate that the existence of prokaryotic nanocompartments extends beyond the sequence homologs of the previously characterized encapsulins and that there exist additional, evolutionarily distinct families of encapsulins with unique cargo systems. We have characterized one of these new encapsulin families, which we name Family 2A, from the cyanobacteria Synechococcus elongatus PCC 7942. This encapsulin system hosts a cysteine desulfurase cargo enzyme that is directed to the interior of the nanocompartment via an N-terminal targeting sequence. We have determined the structure of the Family 2A encapsulin using cryo- electron microscopy to 2.2 Å resolution. This structure has yielded insights into the cargo- binding site within the organelle and the potential size and charge selectivity of the compartment. Additionally, we have characterized the cysteine desulfurase activity of the Family 2A encapsulin and show an increase in cargo enzyme activity upon encapsulation.Finally, we examine the biochemistry and physiological role of a previously discovered encapsulin from Mycobacterium tuberculosis (Mtb). We demonstrate that the Mtb

For many decades, it was thought that the phenomenon of cellular compartmentalization was exclusive to eukaryotes. Advances in cell imaging techniques have revealed that the presence of subcellular compartments is widespread throughout prokaryotes and that there exists a great diversity of organelles in bacteria and archaea. Recently, a new class of prokaryotic organelle has been discovered – the protein-bounded prokaryotic nanocompartments, also called encapsulins. Encapsulins are among the simplest of the prokaryotic organelles, often comprised of a two gene system; a shell-encoding gene and a cargo-encoding gene. Despite their simplicity, little is known about the biochemistry and physiological function of the encapsulins.Here we investigate the structure and function of the encapsulin nanocompartments from various prokaryotes. First, we demonstrate that the existence of prokaryotic nanocompartments extends beyond the sequence homologs of the previously characterized encapsulins and that there exist additional, evolutionarily distinct families of encapsulins with unique cargo systems. We have characterized one of these new encapsulin families, which we name Family 2A, from the cyanobacteria Synechococcus elongatus PCC 7942. This encapsulin system hosts a cysteine desulfurase cargo enzyme that is directed to the interior of the nanocompartment via an N-terminal targeting sequence. We have determined the structure of the Family 2A encapsulin using cryo- electron microscopy to 2.2 Å resolution. This structure has yielded insights into the cargo- binding site within the organelle and the potential size and charge selectivity of the compartment. Additionally, we have characterized the cysteine desulfurase activity of the Family 2A encapsulin and show an increase in cargo enzyme activity upon encapsulation.Finally, we examine the biochemistry and physiological role of a previously discovered encapsulin from Mycobacterium tuberculosis (Mtb). We demonstrate that the Mtb

Xanthine dehydrogenase/oxidase (XDH/XO) is a molybdenum-containing enzyme which is involved with hydroxylation a number of sp2-hybridized centers including purines, heterocycles, and aldehydes. Its main role in the cell is to convert hypoxanthine to xanthine and xanthine to uric acid. Although xanthine oxidase has been studied for decades, details of its mechanism and how the active site allows for substrate specificity and catalysis are still not completely known. In the present work, several methods have been used to investigate the mechanism of xanthine oxidase and the roles of the active site residues towards substrate binding and catalysis. 1.) The kinetic rates of bovine xanthine oxidase and variants of the homologous Rhodobacter capsulatus xanthine dehydrogenase toward various substrates have been observed. Investigation of the E232Q variant of R. capsulatus xanthine dehydrogenase revealed that removal of the ionizable Glutamate 232 resulted in a dramatic loss in activity at higher pH, as compared to wild-type enzyme, providing insight into the role of Glu 232. 2.) We used X-ray crystallography to investigate interactions of the enzyme with the slow substrate indole-3-aldehyde and the non-substrate guanine. The dominant nonproductive orientations of the molecules correlate with the observed kinetic rates. 3.) The effects of active site residues on the chemical step of the reaction were investigated utilizing kinetic isotope effect studies. With the primary deuterium isotope effects, previously described by Dr. Cao, and intrinsic isotope effects Dk, derived from the tritium isotope effect studies conducted for bovine xanthine oxidase and bacterial xanthine dehydrogenase, the extent that the chemical step is rate-limiting was calculated for each. Comparison of the enzymes with amino acid substitution variants allowed for insight into the role of the active site residues by the monitoring of changes (or lack of change) in the rate of the chemical step and it's c

Xanthine dehydrogenase/oxidase (XDH/XO) is a molybdenum-containing enzyme which is involved with hydroxylation a number of sp2-hybridized centers including purines, heterocycles, and aldehydes. Its main role in the cell is to convert hypoxanthine to xanthine and xanthine to uric acid. Although xanthine oxidase has been studied for decades, details of its mechanism and how the active site allows for substrate specificity and catalysis are still not completely known. In the present work, several methods have been used to investigate the mechanism of xanthine oxidase and the roles of the active site residues towards substrate binding and catalysis. 1.) The kinetic rates of bovine xanthine oxidase and variants of the homologous Rhodobacter capsulatus xanthine dehydrogenase toward various substrates have been observed. Investigation of the E232Q variant of R. capsulatus xanthine dehydrogenase revealed that removal of the ionizable Glutamate 232 resulted in a dramatic loss in activity at higher pH, as compared to wild-type enzyme, providing insight into the role of Glu 232. 2.) We used X-ray crystallography to investigate interactions of the enzyme with the slow substrate indole-3-aldehyde and the non-substrate guanine. The dominant nonproductive orientations of the molecules correlate with the observed kinetic rates. 3.) The effects of active site residues on the chemical step of the reaction were investigated utilizing kinetic isotope effect studies. With the primary deuterium isotope effects, previously described by Dr. Cao, and intrinsic isotope effects Dk, derived from the tritium isotope effect studies conducted for bovine xanthine oxidase and bacterial xanthine dehydrogenase, the extent that the chemical step is rate-limiting was calculated for each. Comparison of the enzymes with amino acid substitution variants allowed for insight into the role of the active site residues by the monitoring of changes (or lack of change) in the rate of the chemical step and it's c

Leaf senescence in Arabidopsis thaliana is a strict, genetically controlled nutrient recovery program, which typically progresses in an age-dependent manner. Leaves of the Arabidopsis onset of leaf death5 ( old5) mutant exhibit early developmental senescence. Here, we show that OLD5 encodes quinolinate synthase ( QS), a key enzyme in the de novo synthesis of NAD. The Arabidopsis QS was previously shown to carry a Cys desulfurase domain that stimulates reconstitution of the oxygen-sensitive Fe-S cluster that is required for QS activity. The old5 lesion in this enzyme does not affect QS activity but it decreases its Cys desulfurase activity and thereby the long-term catalytic competence of the enzyme. The old5 mutation causes increased NAD steady state levels that coincide with increased activity of enzymes in the NAD salvage pathway. NAD plays a key role in cellular redox reactions, including those of the tricarboxylic acid cycle. Broad-range metabolite profiling of the old5 mutant revealed that it contains higher levels of tricarboxylic acid cycle intermediates and nitrogen-containing amino acids. The mutant displays a higher respiration rate concomitant with increased expression of oxidative stress markers. We postulate that the alteration in the oxidative state is integrated into the plant developmental program, causing early ageing of the mutant.