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Kynurenic acid (KA) is a tryptophan (Trp) metabolite that is synthesised in a branch of kynurenine (KYN) pathway. KYN aminotransferase (KAT) catalyses deamination of KYN, yielding KA. Although KA synthesis is evolutionarily conserved from bacteria to humans, the cellular benefits of synthesising KA are unclear. In this study, we constructed a KAT-null yeast mutant defective in KA synthesis to clarify the cellular function of KA. Amino acid sequence analysis and LC/MS quantification of KA revealed that Aro8 and Aro9 are the major KATs. KA was significantly decreased in the aro8Δ aro9Δ double mutant. We found that aro8Δ aro9Δ cells did not exhibit obvious defects in growth or oxidative stress response when proper amounts of amino acids are supplied in the media. We further found that aro8Δ aro9Δ cells were sensitive to excess Trp. The Trp sensitivity was not rescued by addition of KA, suggesting that Trp sensitivity is not due to the loss of KA. In conclusion, we propose that KAT activity is required for detoxification of Trp by converting it to the less toxic KA.

Autophagy is an enigmatic cellular process in which double-membrane compartments, called “autophagosomes, form de novo adjacent to the endoplasmic reticulum (ER) and package cytoplasmic contents for delivery to lysosomes. Expansion of the precursor membrane phagophore requires autophagy-related 2 (ATG2), which localizes to the PI3P-enriched ER–phagophore junction. We combined single-particle electron microscopy, chemical cross-linking coupled with mass spectrometry, and biochemical analyses to characterize human ATG2A in complex with the PI3P effector WIPI4. ATG2A is a rod-shaped protein that can bridge neighboring vesicles through interactions at each of its tips. WIPI4 binds to one of the tips, enabling the ATG2A-WIPI4 complex to tether a PI3P-containing vesicle to another PI3P-free vesicle. These data suggest that the ATG2A-WIPI4 complex mediates ER–phagophore association and/or tethers vesicles to the ER–phagophore junction, establishing the required organization for phagophore expansion via the transfer of lipid membranes from the ER and/or the vesicles to the phagophore.

NAD kinase is the sole NADP + biosynthetic enzyme. Despite the great significance of NADP + , to date no mitochondrial NAD kinase has been identified in human, and the source of human mitochondrial NADP + remains elusive. Here we present evidence demonstrating that a human protein of unknown function, C5orf33, is a human mitochondrial NAD kinase; this protein likely represents the missing source of human mitochondrial NADP + . The C5orf33 protein exhibits NAD kinase activity, utilizing ATP or inorganic polyphosphate, and is localized in the mitochondria of human HEK293A cells. C5orf33 mRNA is more abundant than human cytosolic NAD kinase mRNA in almost all tissues examined. We further show by database searches that some animals and protists carry C5orf33 homologues as their sole NADP + biosynthetic enzyme, whereas plants and fungi possess no C5orf33 homologue. These observations provide insights into eukaryotic NADP + biosynthesis, which has pivotal roles in cells and organelles. Despite the central importance of NADP + in mitochondrial energy metabolism, no NAD kinase has been found in human mitochondria to synthesize NADP + . These authors show C5orf33, a protein specific to animals and protists, to be the missing NAD kinase and demonstrate its mitochondrial localization and activity.

Members of the ATG8 family of ubiquitin-like proteins (Ubls) are conjugated to phosphatidylethanolamine (PE) in the autophagosomal membrane, where they recruit degradation substrates and facilitate membrane biogenesis. Despite this well-characterized function, the mechanisms underlying the lipidation process, including the action of the E2 enzyme ATG3, remain incompletely understood. Here, we report the crystal structure of human ATG3 conjugated to the mammalian ATG8 protein GABARAP via an isopeptide bond, mimicking the Ubl∼E2 thioester intermediate. In this structure, the GABARAP∼ATG3 conjugate adopts an open configuration with minimal contacts between the two proteins. Notably, the crystal lattice reveals non-covalent contacts between GABARAP and the backside of ATG3's E2 catalytic center, resulting in the formation of a helical filament of the GABARAP∼ATG3 conjugate. While similar filament formations have been observed with canonical Ub∼E2 conjugates, the E2 backside-binding interface of GABARAP is distinct from those of Ub/Ubl proteins and overlaps with the binding site for LC3 interacting region (LIR) peptides. NMR analysis confirms the presence of this non-covalent interaction in solution, and mutagenesis experiments demonstrate the involvement of the E2 backside in PE conjugation. These findings highlight the critical role of the E2 backside in the lipidation process and suggest evolutionary adaptations in the unique E2 enzyme ATG3.

NAD kinase (NADK, EC 2.7.1.23) is the sole NADP + -biosynthetic enzyme that catalyzes phosphorylation of NAD + to yield NADP + using ATP as a phosphoryl donor, and thus, plays a vital role in the cell and represents a potentially powerful antimicrobial drug target. Although methods for expression and purification of human NADK have been previously established (Lerner et al. Biochem Biophys Res Commun 288:69–74, 2001 ), the purification procedure could be significantly improved. In this study, we improved the method for expression and purification of human NADK in Escherichia coli and obtained a purified homogeneous enzyme only through heat treatment and single column chromatography. Using the purified human NADK, we revealed a sigmoidal kinetic behavior toward ATP and the inhibitory effects of NADPH and NADH, but not of NADP + , on the catalytic activity of the enzyme. These inhibitory effects provide insight into the regulation of intracellular NADPH synthesis. Furthermore, these attributes may provide a clue to design a novel drug against Mycobacterium tuberculosis in which this bacterial NADK is potently inhibited by NADP + .

NAD kinase (NADK, EC 2.7.1.23) is the sole NADP super(+)-biosynthetic enzyme that catalyzes phosphorylation of NAD super(+) to yield NADP super(+) using ATP as a phosphoryl donor, and thus, plays a vital role in the cell and represents a potentially powerful antimicrobial drug target. Although methods for expression and purification of human NADK have been previously established (Lerner et al. Biochem Biophys Res Commun 288:69-74, 2001), the purification procedure could be significantly improved. In this study, we improved the method for expression and purification of human NADK in Escherichia coli and obtained a purified homogeneous enzyme only through heat treatment and single column chromatography. Using the purified human NADK, we revealed a sigmoidal kinetic behavior toward ATP and the inhibitory effects of NADPH and NADH, but not of NADP super(+), on the catalytic activity of the enzyme. These inhibitory effects provide insight into the regulation of intracellular NADPH synthesis. Furthermore, these attributes may provide a clue to design a novel drug against Mycobacterium tuberculosis in which this bacterial NADK is potently inhibited by NADP super(+).

NAD kinase (NADK, EC 2.7.1.23) is the sole [NADP.sup.+]-biosynthetic enzyme that catalyzes phosphorylation of [NAD.sup.+] to yield [NADP.sup.+] using ATP as a phosphoryl donor, and thus, plays a vital role in the cell and represents a potentially powerful antimicrobial drug target. Although methods for expression and purification of human NADK have been previously established (Lerner et al. Biochem Biophys Res Commun 288:69-74, 2001), the purification procedure could be significantly improved. In this study, we improved the method for expression and purification of human NADK in Escherichia coli and obtained a purified homogeneous enzyme only through heat treatment and single column chromatography. Using the purified human NADK, we revealed a sigmoidal kinetic behavior toward ATP and the inhibitory effects of NADPH and NADH, but not of [NADP.sup.+], on the catalytic activity of the enzyme. These inhibitory effects provide insight into the regulation of intracellular NADPH synthesis. Furthermore, these attributes may provide a clue to design a novel drug against Mycobacterium tuberculosis in which this bacterial NADK is potently inhibited by [NADP.sup.+]. Keywords NAD kinase * Human * Drug design * [NADP.sup.+] NADPH NADH

NAD kinase (NADK, EC 2.7.1.23) is the sole NADP^sup +^-biosynthetic enzyme that catalyzes phosphorylation of NAD^sup +^ to yield NADP^sup +^ using ATP as a phosphoryl donor, and thus, plays a vital role in the cell and represents a potentially powerful antimicrobial drug target. Although methods for expression and purification of human NADK have been previously established (Lerner et al. Biochem Biophys Res Commun 288:69-74, 2001), the purification procedure could be significantly improved. In this study, we improved the method for expression and purification of human NADK in Escherichia coli and obtained a purified homogeneous enzyme only through heat treatment and single column chromatography. Using the purified human NADK, we revealed a sigmoidal kinetic behavior toward ATP and the inhibitory effects of NADPH and NADH, but not of NADP^sup +^, on the catalytic activity of the enzyme. These inhibitory effects provide insight into the regulation of intracellular NADPH synthesis. Furthermore, these attributes may provide a clue to design a novel drug against Mycobacterium tuberculosis in which this bacterial NADK is potently inhibited by NADP^sup +^.[PUBLICATION ABSTRACT]

Autophagy is an enigmatic cellular process in which double-membrane compartments, called autophagosomes, form de novo adjacent to the endoplasmic reticulum (ER) and package cytoplasmic contents for delivery to lysosomes. Expansion of the precursor membrane phagophore requires autophagy-related 2 (ATG2), which localizes to the phosphatidylinositol-3-phosphate (PI3P)-enriched ER-phagophore junction. We combined single-particle electron microscopy, chemical cross-linking coupled with mass spectrometry, and biochemical analyses to characterize human ATG2A in complex with the PI3P effector WIPI4. ATG2A is a rod-shaped protein that can bridge neighboring vesicles through interactions at each of its tips. WIPI4 binds to one of the tips, enabling the ATG2A-WIPI4 complex to tether a PI3P-containing vesicle to another PI3P-free vesicle. These data suggest that the ATG2A-WIPI4 complex mediates ER-phagophore association and/or tethers vesicles to the ER-phagophore junction, establishing the required organization for phagophore expansion via the transfer of lipid membranes from the ER and/or the vesicles to the phagophore. NOTE: Gabriel C. Lander and Takanori Otomo are both co-corresponding authors. Footnotes * The title and the contents have been revised.

Autophagy is an enigmatic cellular process in which double-membrane compartments, called autophagosomes, form de novo adjacent to the endoplasmic reticulum (ER) and package cytoplasmic contents for delivery to lysosomes. Expansion of the precursor membrane phagophore requires autophagy-related 2 (ATG2), which localizes to the phosphatidylinositol-3-phosphate (PI3P)-enriched ER-phagophore junction. We combined single-particle electron microscopy, chemical cross-linking coupled with mass spectrometry, and biochemical analyses to characterize human ATG2A in complex with the PI3P effector WIPI4. ATG2A is a rod-shaped protein that can bridge neighboring vesicles through interactions at each of its tips. WIPI4 binds to one of the tips, enabling the ATG2A-WIPI4 complex to tether a PI3P-containing vesicle to another PI3P-free vesicle. These data suggest that the ATG2A-WIPI4 complex mediates ER-phagophore association and/or tethers vesicles to the ER-phagophore junction, establishing the required organization for phagophore expansion via the transfer of lipid membranes from the ER and/or the vesicles to the phagophore.