Nrt1 and Tna1-Independent Export of NAD+ Precursor Vitamins Promotes NAD+ Homeostasis and Allows Engineering of Vitamin Production

NAD+ is both a co-enzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD+ consuming enzymes. NAD+ biosynthesis is required for two different regimens that extend lifespan in yeast. NAD+ is synthesized from tryptophan and the three vitamin precursors of NAD+: nicotinic acid, nicotinamide and nicotinamide riboside. Supplementation of yeast cells with NAD+ precursors increases intracellular NAD+ levels and extends replicative lifespan. Here we show that both nicotinamide riboside and nicotinic acid are not only vitamins but are also exported metabolites. We found that the deletion of the nicotinamide riboside transporter, Nrt1, leads to increased export of nicotinamide riboside. This discovery was exploited to engineer a strain to produce high levels of extracellular nicotinamide riboside, which was recovered in purified form. We further demonstrate that extracellular nicotinamide is readily converted to extracellular nicotinic acid in a manner that requires intracellular nicotinamidase activity. Like nicotinamide riboside, export of nicotinic acid is elevated by the deletion of the nicotinic acid transporter, Tna1. The data indicate that NAD+ metabolism has a critical extracellular element in the yeast system and suggest that cells regulate intracellular NAD+ metabolism by balancing import and export of NAD+ precursor vitamins

Glutamine versus Ammonia Utilization in the NAD Synthetase Family

NAD is a ubiquitous and essential metabolic redox cofactor which also functions as a substrate in certain regulatory pathways. The last step of NAD synthesis is the ATP-dependent amidation of deamido-NAD by NAD synthetase (NADS). Members of the NADS family are present in nearly all species across the three kingdoms of Life. In eukaryotic NADS, the core synthetase domain is fused with a nitrilase-like glutaminase domain supplying ammonia for the reaction. This two-domain NADS arrangement enabling the utilization of glutamine as nitrogen donor is also present in various bacterial lineages. However, many other bacterial members of NADS family do not contain a glutaminase domain, and they can utilize only ammonia (but not glutamine) in vitro. A single-domain NADS is also characteristic for nearly all Archaea, and its dependence on ammonia was demonstrated here for the representative enzyme from Methanocaldococcus jannaschi. However, a question about the actual in vivo nitrogen donor for single-domain members of the NADS family remained open: Is it glutamine hydrolyzed by a committed (but yet unknown) glutaminase subunit, as in most ATP-dependent amidotransferases, or free ammonia as in glutamine synthetase? Here we addressed this dilemma by combining evolutionary analysis of the NADS family with experimental characterization of two representative bacterial systems: a two-subunit NADS from Thermus thermophilus and a single-domain NADS from Salmonella typhimurium providing evidence that ammonia (and not glutamine) is the physiological substrate of a typical single-domain NADS. The latter represents the most likely ancestral form of NADS. The ability to utilize glutamine appears to have evolved via recruitment of a glutaminase subunit followed by domain fusion in an early branch of Bacteria. Further evolution of the NADS family included lineage-specific loss of one of the two alternative forms and horizontal gene transfer events. Lastly, we identified NADS structural elements associated with glutamine-utilizing capabilities

E. coli Histidine Triad Nucleotide Binding Protein 1 (ecHinT) Is a Catalytic Regulator of D-Alanine Dehydrogenase (DadA) Activity In Vivo

Histidine triad nucleotide binding proteins (Hints) are highly conserved members of the histidine triad (HIT) protein superfamily. Hints comprise the most ancient branch of this superfamily and can be found in Archaea, Bacteria, and Eukaryota. Prokaryotic genomes, including a wide diversity of both Gram-negative and Gram-positive bacteria, typically have one Hint gene encoded by hinT (ycfF in E. coli). Despite their ubiquity, the foundational reason for the wide-spread conservation of Hints across all kingdoms of life remains a mystery. In this study, we used a combination of phenotypic screening and complementation analyses with wild-type and hinT knock-out Escherichia coli strains to show that catalytically active ecHinT is required in E. coli for growth on D-alanine as a sole carbon source. We demonstrate that the expression of catalytically active ecHinT is essential for the activity of the enzyme D-alanine dehydrogenase (DadA) (equivalent to D-amino acid oxidase in eukaryotes), a necessary component of the D-alanine catabolic pathway. Site-directed mutagenesis studies revealed that catalytically active C-terminal mutants of ecHinT are unable to activate DadA activity. In addition, we have designed and synthesized the first cell-permeable inhibitor of ecHinT and demonstrated that the wild-type E. coli treated with the inhibitor exhibited the same phenotype observed for the hinT knock-out strain. These results reveal that the catalytic activity and structure of ecHinT is essential for DadA function and therefore alanine metabolism in E. coli. Moreover, they provide the first biochemical evidence linking the catalytic activity of this ubiquitous protein to the biological function of Hints in Escherichia coli

Budding Yeast Dma Proteins Control Septin Dynamics and the Spindle Position Checkpoint by Promoting the Recruitment of the Elm1 Kinase to the Bud Neck

The first step towards cytokinesis in budding yeast is the assembly of a septin ring at the future site of bud emergence. Integrity of this ring is crucial for cytokinesis, proper spindle positioning, and the spindle position checkpoint (SPOC). This checkpoint delays mitotic exit and cytokinesis as long as the anaphase spindle does not properly align with the division axis. SPOC signalling requires the Kin4 protein kinase and the Kin4-regulating Elm1 kinase, which also controls septin dynamics. Here, we show that the two redundant ubiquitin-ligases Dma1 and Dma2 control septin dynamics and the SPOC by promoting the efficient recruitment of Elm1 to the bud neck. Indeed, dma1 dma2 mutant cells show reduced levels of Elm1 at the bud neck and Elm1-dependent activation of Kin4. Artificial recruitment of Elm1 to the bud neck of the same cells is sufficient to re-establish a normal septin ring, proper spindle positioning, and a proficient SPOC response in dma1 dma2 cells. Altogether, our data indicate that septin dynamics and SPOC function are intimately linked and support the idea that integrity of the bud neck is crucial for SPOC signalling

Compositional microprogram control unit with code sharing and control microinstructions

W artykule przedstawiona została metoda syntezy umożliwiająca zmniejszenie liczby tablic LUT potrzebnych do realizacji układu mikroprogramowanego z współdzieleniem kodów. Metoda jest przeznaczona dla układów FPGA z osadzonymi blokami pamięci. Część kombinacyjna układu mikroprogramowanego jest realizowana z użyciem tablic LUT, natomiast pamięć sterująca z użyciem osadzonych bloków pamięci. Redukcję liczby tablic LUT osiągnięto dzięki wykorzystaniu klas łańcuchów pseudorównoważnych. W artykule przedstawiono przykład zastosowania proponowanej metody oraz rezultaty eksperymentów.The paper presents new research results of synthesis of Composi-tional Microprogram Control Unit (CMCU) with Codes Sharing. The method allows reduction of look-up table elements in the combina-tional part of the control unit. The method assumes application of field-programmable gate arrays for implementation of the combinational part, whereas embedded-memory blocks are used for implementation of its control memory. Programmable logic devices are nowadays widely used for implementation of Control Units (CU) [16, 18]. The problem of the CU optimisation is still actual in computer science and it solution permits to decrease the cost of the system [17]. The proposed method is oriented on reduction of hardware amount of CMCU addressing circuit by placing codes of classes of pseudoequivalent states in the control memory. These classes are formed by division of the set of Operational Linear Chains (OLC) into partitions which correspond to pseudoequivalent states of Moore FSM [4]. The research results show that application of the method to tested control algorithms gives on average 50% decrease in hardware amount when compared to CMCU based structure (Tab. 2). The results were obtained using Xilinx ISE. The models of control units were generated by the authors' software using the control algorithms from [15]

Hardware implementation of MD5 algorithm in FPGAs using compositional microprogram control unit

W artykule przedstawiona została koncepcja implementacji sprzętowej algorytmu MD5 z wykorzystaniem mikroprogramowanego układu sterującego. Cechą charakterystyczną rozwiązania jest wykorzystanie osadzonych bloków pamięci do realizacji układu sterującego. Przedstawione rozwiązanie jest przeznaczone przede wszystkim do realizacji w układach FPGA. W artykule przedstawione zostały wyniki syntezy kilku wybranych struktur układów mikroprogramowanych. Otrzymane wyniki zostały porównane do typowej realizacji w postaci automatu Moore'a.The paper presents an example of application of Compositional Microprogram Control Unit (CMCU) to hardware implementation of MD5 algorithm. The MD5 algorithm is a widely used hash function with a 128-bit hash value. MD5 is used in many security applications, for example to hash passwords in FreeBSD operating system [14]. MD5 is also commonly used to check the integrity of files. MD5 was designed by Ron Rivest in 1991 [10]. Other similar algorithms are SHA [7] and RIPEMD [6]. The hardware implementation of MD5 in FPGAs is usually based on embedded memory blocks (EMB) because the algorithm uses a lot of constants during calculations [8]. In the paper the authors present an alternative solution in which constants are generated by CMCU (Fig. 3) circuit. The CMCU is also based on EMB. It can generate constants for MD5 and also signals for other tasks. The research results show that CMCU requires less hardware amount when compared to traditional Moore FSM (Tab. 1). The results were obtained using Xilinx ISE 12.1 and Xilinx Spartan-3 (xc3s50-5pq208) [13]. The models of control units were generated by the authors' software