Identification and characterization of a tyramine-glutamate ligase (MfnD) involved in methanofuran biosynthesis.

Methanofuran is the first in a series of coenzymes involved in the reduction of carbon dioxide to methane. All methanofuran structural variants contain a basic core structure of 4-[N-(γ-l-glutamyl-γ-l-glutamyl)-p-(ß-aminoethyl)phenoxymethyl]-2-(aminomethyl)furan (APMF-(Glu)2) with different attached side chains depending on the source organism. Recently, we discovered the biosynthetic route for the production of 5-(aminomethyl)-3-furanmethanol-phosphate (F1-P), a precursor to the furan moiety of methanofuran. However, how the γ-linked glutamates are incorporated into methanofuran's structure remains unknown. Here, we report the identification of an ATP-grasp enzyme encoded by the gene Mefer_1180 in Methanocaldococcus fervens (the homologue of MJ0815 in Methanocaldococcus jannaschii, annotated as MfnD) that catalyzes the ATP-dependent addition of one glutamate to tyramine via a γ-linked amide bond. The occurrence of this reaction is consistent with the presence of γ-glutamyltyramine in cell extracts of M. jannaschii. Our steady-state kinetic analysis of the recombinant enzyme showed that MfnD exhibits a catalytic ability comparable to other ATP-grasp enzymes such as the Escherichia coli glutathione synthetase (GS), with a similar apparent kcat and KM. In addition, its activity is divalent metal-dependent, with the highest activity observed with Mn(2+). The previously solved crystal structure of MfnD from Archaeoglobus fulgidus exhibits a classical ATP-grasp fold with three structural domains; the ATP-binding and metal-binding motifs are conserved in MfnD as seen in other ATP-grasp enzymes. We used site-directed mutagenesis and kinetic analysis to demonstrate that Arg251 is an important residue for both catalysis and glutamate binding. By comparing the active site of MfnD with GS and by molecular docking substrates to the MfnD active site, we predicted the possible glutamate- and tyramine-binding pocket. This is the first report describing the enzymology of the incorporation of the initial l-glutamate molecule into the methanofuran structure. It also provides the first example of an ATP-grasp enzyme activating the γ-carboxylate of glutamate as substrate.

Steroidal glycoalkaloids in Solanum chacoense.

Potato (Solanum tuberosum L.), a domesticated species that is the fourth most important world agricultural commodity, requires significant management to minimize the effects of herbivore and pathogen damage on crop yield. A wild relative, Solanum chacoense Bitt., has been of interest to plant breeders because it produces an abundance of novel steroidal glycoalkaloid compounds, leptines and leptinines, which are particularly effective deterrents of herbivory by the Colorado potato beetle (Leptinotarsa decemlineata Say). Biochemical approaches were used in this study to investigate the formation and accumulation of SGAs in S. chacoense. SGA contents were determined in various organs at different stages of organ maturity during a time course of plant development. Leptines and leptinines were the main contributors to the increased levels in SGA concentration measured in the aerial versus the subterranean organs of S. chacoense accession 8380-1. Leptines were not detected in aboveground stolons until the stage where shoots had formed mature chlorophyllous leaves. To gain insights into SGA biosynthesis, the abundance of SGAs and steady-state transcripts of genes coding for enzymes of the central terpene and SGA-specific pathways in various plant organs at anthesis were compared. For two genes of primary terpene metabolism, transcript and SGA abundances were correlated, although with some discrepancies. For genes associated with SGA biosynthesis, transcripts were not detected in some tissues containing SGAs; however these transcripts were detected in the progenitor tissues, indicating the possibility that under our standard growth conditions, SGA biosynthesis is largely limited to highly proliferative tissues such as shoot, root and floral meristems.

Tobacco nicotine uptake permease (NUP1) affects alkaloid metabolism.

An effective plant alkaloid chemical defense requires a variety of transport processes, but few alkaloid transporters have been characterized at the molecular level. Previously, a gene fragment encoding a putative plasma membrane proton symporter was isolated, because it was coordinately regulated with several nicotine biosynthetic genes. Here, we show that this gene fragment corresponds to a Nicotiana tabacum gene encoding a nicotine uptake permease (NUP1). NUP1 belongs to a plant-specific class of purine uptake permease-like transporters that originated after the bryophytes but before or within the lycophytes. NUP1 expressed in yeast cells preferentially transported nicotine relative to other pyridine alkaloids, tropane alkaloids, kinetin, and adenine. NUP1-GFP primarily localized to the plasma membrane of tobacco Bright Yellow-2 protoplasts. WT NUP1 transcripts accumulated to high levels in the roots, particularly in root tips. NUP1-RNAi hairy roots had reduced NUP1 mRNA accumulation levels, reduced total nicotine levels, and increased nicotine accumulation in the hairy root culture media. Regenerated NUP1-RNAi plants showed reduced foliar and root nicotine levels as well as increased seedling root elongation rates. Thus, NUP1 affected nicotine metabolism, localization, and root growth.

Shiga-like toxin subunit B (SLTB)-enhanced delivery of chlorin e6 (Ce6) improves cell killing.

We used Shiga-like toxin B subunit (SLTB) to deliver the photosensitizer, chlorin e6 (Ce6), to Vero cells expressing the Gb3 receptor. Our aim was to provide an example of carrier-enhanced photodynamic cell killing with which to start a systematic consideration of photosensitizer delivery at the subcellular level. SLTB, in contrast to many other potential protein carriers, is delivered intracellularly to the Golgi apparatus and endoplasmic reticulum (ER). Ce6 was chosen both for its phototoxic properties and its potential for covalent conjugation with SLTB. Ce6-SLTB after cleanup contained < or =10% noncovalently bound Ce6. The noncovalent binding of porphyrins and chlorins to protein conjugates has been well documented, and hence the effective cleanup procedure is a significant accomplishment. We demonstrate that Ce6-SLTB enhances delivery of Ce6 to target cells as compared to free Ce6. In Vero cells, Ce6-SLTB was over an order of magnitude more photodynamically toxic than free Ce6. Moreover, we show that in the case of Ce6-SLTB, photosensitizer accumulation is in a combination of subcellular sites including mitochondria, Golgi apparatus, ER and plasma membrane. The occurrence in nature of diverse B subunit binding sites and the possibilities of varied intracellular delivery make optimized use of B subunit carriers attractive.