Identification of a metabolic disposal route for the oncometabolite S-(2-succino)cysteine in Bacillus subtilis.

Cellular thiols such as cysteine spontaneously and readily react with the respiratory intermediate fumarate, resulting in the formation of stable S-(2-succino)-adducts. Fumarate-mediated succination of thiols increases in certain tumors and in response to glucotoxicity associated with diabetes. Therefore, S-(2-succino)-adducts such as S-(2-succino)cysteine (2SC) are considered oncometabolites and biomarkers for human disease. No disposal routes for S-(2-succino)-compounds have been reported prior to this study. Here, we show that Bacillus subtilis metabolizes 2SC to cysteine using a pathway encoded by the yxe operon. The first step is N-acetylation of 2SC followed by an oxygenation that we propose results in the release of oxaloacetate and N-acetylcysteine, which is deacetylated to give cysteine. Knockouts of the genes predicted to mediate each step in the pathway lose the ability to grow on 2SC as the sulfur source and accumulate the expected upstream metabolite(s). We further show that N-acetylation of 2SC relieves toxicity. This is the first demonstration of a metabolic disposal route for any S-(2-succino)-compound, paving the way toward the identification of corresponding pathways in other species.

A plastidial pantoate transporter with a potential role in pantothenate synthesis.

The pantothenate (vitamin B5) synthesis pathway in plants is not fully defined because the subcellular site of its ketopantoate → pantoate reduction step is unclear. However, the pathway is known to be split between cytosol, mitochondria, and potentially plastids, and inferred to involve mitochondrial or plastidial transport of ketopantoate or pantoate. No proteins that mediate these transport steps have been identified. Comparative genomic and transcriptomic analyses identified Arabidopsis thaliana BASS1 (At1g78560) and its maize (Zea mays) ortholog as candidates for such a transport role. BASS1 proteins belong to the bile acid : sodium symporter family and share similarity with the Salmonella enterica PanS pantoate/ketopantoate transporter and with predicted bacterial transporters whose genes cluster on the chromosome with pantothenate synthesis genes. Furthermore, Arabidopsis BASS1 is co-expressed with genes related to metabolism of coenzyme A, the cofactor derived from pantothenate. Expression of Arabidopsis or maize BASS1 promoted the growth of a S. enterica panB panS mutant strain when pantoate, but not ketopantoate, was supplied, and increased the rate of [3H]pantoate uptake. Subcellular localization of green fluorescent protein fusions in Nicotiana tabacum BY-2 cells demonstrated that Arabidopsis BASS1 is targeted solely to the plastid inner envelope. Two independent Arabidopsis BASS1 knockout mutants accumulated pantoate ∼10-fold in leaves and had smaller seeds. Taken together, these data indicate that BASS1 is a physiologically significant plastidial pantoate transporter and that the pantoate reduction step in pantothenate biosynthesis could be at least partly localized in plastids.

Essential role of conserved DUF177A protein in plastid 23S rRNA accumulation and plant embryogenesis.

DUF177 proteins are nearly universally conserved in bacteria and plants except the Chlorophyceae algae. Thus far, duf177 mutants in bacteria have not established a function. In contrast, duf177a mutants have embryo lethal phenotypes in maize and Arabidopsis. In maize inbred W22, duf177a mutant embryos arrest at an early transition stage, whereas the block is suppressed in the B73 inbred background, conditioning an albino seedling phenotype. Background-dependent embryo lethal phenotypes are characteristic of maize plastid gene expression mutants. Consistent with the plastid gene expression hypothesis, quantitative real-time PCR revealed a significant reduction of 23S rRNA in an Escherichia coli duf177 knockout. Plastid 23S rRNA contents of duf177a mutant tissues were also markedly reduced compared with the wild-type, whereas plastid 16S, 5S, and 4.5S rRNA contents were less affected, indicating that DUF177 is specifically required for accumulation of prokaryote-type 23S rRNA. An AtDUF177A-green fluorescent protein (GFP) transgene controlled by the native AtDUF177A promoter fully complemented the Arabidopsis atduf177a mutant. Transient expression of AtDUF177A-GFP in Nicotiana benthamiana leaves showed that the protein was localized in chloroplasts. The essential role of DUF177A in chloroplast-ribosome formation is reminiscent of IOJAP, another highly conserved ribosome-associated protein, suggesting that key mechanisms controlling ribosome formation in plastids evolved from non-essential pathways for regulation of the prokaryotic ribosome.

Salvage of the thiamin pyrimidine moiety by plant TenA proteins lacking an active-site cysteine.

The TenA protein family occurs in prokaryotes, plants and fungi; it has two subfamilies, one (TenA_C) having an active-site cysteine, the other (TenA_E) not. TenA_C proteins participate in thiamin salvage by hydrolysing the thiamin breakdown product amino-HMP (4-amino-5-aminomethyl-2-methylpyrimidine) to HMP (4-amino-5-hydroxymethyl-2-methylpyrimidine); the function of TenA_E proteins is unknown. Comparative analysis of prokaryote and plant genomes predicted that (i) TenA_E has a salvage role similar to, but not identical with, that of TenA_C and (ii) that TenA_E and TenA_C also have non-salvage roles since they occur in organisms that cannot make thiamin. Recombinant Arabidopsis and maize TenA_E proteins (At3g16990, GRMZM2G080501) hydrolysed amino-HMP to HMP and, far more actively, hydrolysed the N-formyl derivative of amino-HMP to amino-HMP. Ablating the At3g16990 gene in a line with a null mutation in the HMP biosynthesis gene ThiC prevented its rescue by amino-HMP. Ablating At3g16990 in the wild-type increased sensitivity to paraquat-induced oxidative stress; HMP overcame this increased sensitivity. Furthermore, the expression of TenA_E and ThiC genes in Arabidopsis and maize was inversely correlated. These results indicate that TenA_E proteins mediate amidohydrolase and aminohydrolase steps in the salvage of thiamin breakdown products. As such products can be toxic, TenA_E proteins may also pre-empt toxicity.

The potato tuber, maize endosperm and a chimeric maize-potato ADP-glucose pyrophosphorylase exhibit fundamental differences in Pi inhibition.

ADP-glucose pyrophosphorylase (AGPase) is highly regulated by allosteric effectors acting both positively and negatively. Enzymes from various sources differ, however, in the mechanism of allosteric regulation. Here, we determined how the effector, inorganic phosphate (Pi), functions in the presence and absence of saturating amounts of the activator, 3-phosphoglyceric acid (3-PGA). This regulation was examined in the maize endosperm enzyme, the oxidized and reduced forms of the potato tuber enzyme as well as a small subunit chimeric AGPase (MP), which contains both maize endosperm and potato tuber sequences paired with a wild-type maize large subunit. These data, combined with our previous kinetic studies of these enzymes led to a model of Pi inhibition for the various enzymes. The Pi inhibition data suggest that while the maize enzyme contains a single effector site that binds both 3-PGA and Pi, the other enzymes exhibit more complex behavior and most likely have at least two separate interacting binding sites for Pi. The possible physiological implications of the differences in Pi inhibition distinguishing the maize endosperm and potato tuber AGPases are discussed.

Cellulose Synthase-Like D1 is integral to normal cell division, expansion, and leaf development in maize.

The Cellulose Synthase-Like D (CslD) genes have important, although still poorly defined, roles in cell wall formation. Here, we show an unexpected involvement of CslD1 from maize (Zea mays) in cell division. Both division and expansion were altered in the narrow-organ and warty phenotypes of the csld1 mutants. Leaf width was reduced by 35%, due mainly to a 47% drop in the number of cell files across the blade. Width of other organs was also proportionally reduced. In leaf epidermis, the deficiency in lateral divisions was only partially compensated by a modest, uniform increase in cell width. Localized clusters of misdivided epidermal cells also led to the formation of warty lesions, with cell clusters bulging from the epidermal layer, and some cells expanding to volumes 75-fold greater than normal. The decreased cell divisions and localized epidermal expansions were not associated with detectable changes in the cell wall composition of csld1 leaf blades or epidermal peels, yet a greater abundance of thin, dense walls was indicated by high-resolution x-ray tomography of stems. Cell-level defects leading to wart formation were traced to sites of active cell division and expansion at the bases of leaf blades, where cytokinesis and cross-wall formation were disrupted. Flow cytometry confirmed a greater frequency of polyploid cells in basal zones of leaf blades, consistent with the disruption of cytokinesis and/or the cell cycle in csld1 mutants. Collectively, these data indicate a previously unrecognized role for CSLD activity in plant cell division, especially during early phases of cross-wall formation.