NAD-binding Site Research Articles

Characterization of the Structural Requirements for the NADase Activity of Bacterial Toll/IL-1R domains in a Course-based Undergraduate Research Experience.

TLRs initiate innate immune signaling pathways via Toll/IL-1R (TIR) domains on their cytoplasmic tails. Various bacterial species also express TIR domain-containing proteins that contribute to bacterial evasion of the innate immune system. Bacterial TIR domains, along with the mammalian sterile α and TIR motif-containing protein 1 and TIRs from plants, also have been found to exhibit NADase activity. Initial X-ray crystallographic studies of the bacterial TIR from Acinetobacter baumannii provided insight into bacterial TIR structure but were unsuccessful in cocrystallization with the NAD+ ligand, leading to further questions about the TIR NAD binding site. In this study, we designed a Course-Based Undergraduate Research Experience (CURE) involving 16-20 students per year to identify amino acids crucial for NADase activity of A. baumannii TIR domain protein and the TIR from Escherichia coli (TIR domain-containing protein C). Students used structural data to identify amino acids that they hypothesized would play a role in TIR NADase activity, and created plasmids to express mutated TIRs through site-directed mutagenesis. Mutant TIRs were expressed, purified, and tested for NADase activity. The results from these studies provide evidence for a conformational change upon NAD binding, as was predicted by recent cryogenic electron microscopy and hydrogen-deuterium exchange mass spectrometry studies. Along with corroborating recent characterization of TIR NADases that could contribute to drug development for diseases associated with dysregulated TIR activity, this work also highlights the value of CURE-based projects for inclusion of a diverse group of students in authentic research experiences.

Results from a CURE course project reveal novel insights into the structure of bacterial Toll/interleukin-1 receptor (TIR) domain proteins

Abstract Toll-like receptors (TLRs) are critical for innate immune signaling, and various bacteria express Toll/interleukin-1 receptor (TIR) domains to evade this signaling. Bacterial TIRs exhibit NADase activity, although it is unclear what role NADase activity plays in evasion of innate responses. To better characterize the NADase activity of the TIR from Acenitobacter baumanii (AbTIR), we used site directed mutagenesis to identify amino acids predicted to be important for NADase activity. Studies were carried out in a Course-Based Undergraduate Research Experience (CURE) involving 16–20 undergraduate research students per year. Students used Chimera to analyze the AbTIR structure and hypothesize amino acids they predicted would play a role in enzymatic activity. They then performed site-directed mutagenesis to create plasmids for expression of mutant AbTIR. Mutant TIRs were expressed, purified and tested for NADase activity. So far, our mutagenesis results suggest a potential conformational change in the NAD binding site upon NAD binding. Identifying amino acids critical for enzymatic activity may inform drug development for diseases associated with dysregulated TIR activity. In addition, the application of the CURE class to carry out these studies has provided project-based research training experiences for approximately 80 undergraduate students, about half of whom are members of groups traditionally underrepresented in science. Several students, including some who had limited research experience before completing the course, are currently pursuing graduate studies and research-based careers, highlighting the value of CURE-based projects for facilitating the inclusion of students from diverse groups in research. TVS and TO were supported by the NIH Bridges to Baccalaureate Grant R25GM058264; CP was supported by the NIH Bridges to the Doctorate Grant R25GM119970, and the CURE course was supported by an HHMI Inclusive Excellence Grant

In silico protein engineering shows that novel mutations affecting NAD+ binding sites may improve phosphite dehydrogenase stability and activity

Pseudomonas stutzeri phosphite dehydrogenase (PTDH) catalyzes the oxidation of phosphite to phosphate in the presence of NAD, resulting in the formation of NADH. The regeneration of NADH by PTDH is greater than any other enzyme due to the substantial change in the free energy of reaction (G°′ = − 63.3 kJ/mol). Presently, improving the stability of PTDH is for a great importance to ensure an economically viable reaction process to produce phosphite as a byproduct for agronomic applications. The binding site of NAD+ with PTDH includes thirty-four residues; eight of which have been previously mutated and characterized for their roles in catalysis. In the present study, the unexplored twenty-six key residues involved in the binding of NAD+ were subjected to in silico mutagenesis based on the physicochemical properties of the amino acids. The effects of these mutations on the structure, stability, activity, and interaction of PTDH with NAD+ were investigated using molecular docking, molecular dynamics simulations, free energy calculations, and secondary structure analysis. We identified seven novel mutations, A155I, G157I, L217I, P235A, V262I, I293A, and I293L, that reduce the compactness of the protein while improving PTDH stability and binding to NAD+.

Cloning and in silico characterization of cinnamyl alcohol dehydrogenase gene involved in lignification of Tall fescue (Festuca arundinacea Schreb.).

Tall fescue, a promising temperate forage grass of Himalayan region, possesses extraordinary property of rapid growth with high biomass production, but its poor digestibility due to higher lignin content limits its utilization in livestock feeding. The lignification in Tall fescue is under the control of enzymatic cascade of different regulatory enzymes. Cinnamyl alcohol dehydrogenase (CAD) is a crucial regulatory enzyme that catalyzes the last step of monolignol biosynthesis and is a potential candidate for altering the content and types of lignin, and hence increasing the digestibility of fodder crops. Hence, the present investigation was conducted on isolation, cloning and characterization of CAD gene from Tall fescue. Isolation and amplification of CAD gene resulted in an amplicon of 1521bp. The CAD gene sequence was submitted to NCBI database with an accession number MW442831. Translation of the CAD gene sequence exhibited an ORF of 361 amino acids. The deduced CAD protein was predicted to be hydrophobic, acidic and thermally stable with molecular formula C1712H2734N460O520S23, molecular mass of 38.82kDa, theoretical pI of 5.60 and 3 strong transmembrane helices. The CAD protein was predicted to have a dimer forming behavior with putative NAD(P) binding site between amino acids 48 and 301, putative substrate-binding site between amino acids 48 and 301, catalytic zinc-binding site between amino acids 48 and 164 and structural zinc-binding site between amino acid residue 101 and 115. A conserved 189GLGGVG194 motif is the binding site for NADP(H). The conserved motif pattern of CAD's zinc catalytic center was found to be 69GHEVVGEV(X)EVG(X)2V83. The zinc-binding site was found to be conserved between amino acid 89 and 115 and was found to be 89G(X)2VG(X)G(X)2VGXC(X)2C(X)2C(X)5QYC115. The deciphered sequence and putative protein information might be useful in subsequent research in lignin bioengineering for enhanced digestibility, biomass conversion as well as impact of lignin on cell wall mechanics.

Differential expressions and enzymatic properties of malate dehydrogenases in response to nutrient and metal stresses in Stylosanthes guianensis

Malate dehydrogenase (MDH, EC 1.1.1.37) is a key enzyme that catalyzes a reversible NAD-dependent dehydrogenase reaction from oxaloacetate (OAA) to malate. Although MDH has been documented to participate in cellular metabolism and redox homeostasis in plants, the roles of MDH members in the tropical legume Stylosanthes guianensis (stylo) remain less definitive. In this study, except SgMDH1 that had been previously characterized, six novel MDH genes were isolated from stylo and were then designated as SgMDH2 to SgMDH7. All of the SgMDH proteins possessed the common features of NAD binding, dimerization interface and substrate binding sites. Expression analysis showed that three SgMDHs exhibited preferential expressions in leaves, and one SgMDH was mainly expressed in roots. Furthermore, SgMDHs were regulated by nutrient deficiencies in stylo roots, especially for phosphorus (-P) and potassium (-K) deficiencies. Differential responses of SgMDHs to trace metal stress and heavy metal toxicity were observed in stylo roots, suggesting the involvement of SgMDHs in the response of stylo to metal stresses. The six novel SgMDHs were subsequently expressed and purified from Escherichia coli to analyze their biochemical properties. Although SgMDHs exhibited variations in subcellular localizations, each SgMDH protein displayed a high level of catalytic efficiency towards OAA and NADH but a low level of catalytic efficiency towards malate and NAD+. In addition, the activities of recombinant SgMDH proteins were pH-dependent and temperature-sensitive, and exhibited differential regulations by various metal ions. These results together suggest the potential roles of SgMDHs in stylo coping with nutrient and metal stresses.

Mycophenolic anilides as broad specificity inosine-5’-monophosphate dehydrogenase (IMPDH) inhibitors

Inosine-5′-monophosphate dehydrogenase (IMPDH) is a potential target for microorganisms. However, identifying inhibitor design determinants for IMPDH orthologs continues to evolve. Herein, a series of mycophenolic anilide inhibitors of Cryptosporidium parvum and human IMPDHs are reported. Furthermore, molecular docking of 12 (e.g. SH-19; CpIMPDH Ki,app = 0.042 ± 0.015 µM, HsIMPDH2 Ki,app = 0.13 ± 0.05 µM) supports different binding modes with the two enzymes. For CpIMPDH the inhibitor extends into a pocket in an adjacent subunit. In contrast, docking suggests the inhibitor interacts with Ser276 in the NAD binding site in HsIMPDH2, as well as an adjacent pocket within the same subunit. These results provide further guidance for generating IMPDH inhibitors for enzymes found in an array of pathogenic microorganisms, including Mycobacterium tuberculosis.

Expression, purification and crystal structure determination of a ferredoxin reductase from the actinobacterium Thermobifida fusca.

The ferredoxin reductase FdR9 from Thermobifida fusca, a member of the oxygenase-coupled NADH-dependent ferredoxin reductase (FNR) family, catalyses electron transfer from NADH to its physiological electron acceptor ferredoxin. It forms part of a putative three-component cytochrome P450 monooxygenase system in T. fusca comprising CYP222A1 and the [3Fe-4S]-cluster ferredoxin Fdx8 as well as FdR9. Here, FdR9 was overexpressed and purified and its crystal structure was determined at 1.9 Å resolution. The overall structure of FdR9 is similar to those of other members of the FNR family and is composed of an FAD-binding domain, an NAD-binding domain and a C-terminal domain. Activity measurements with FdR9 confirmed a strong preference for NADH as the cofactor. Comparison of the FAD- and NAD-binding domains of FdR9 with those of other ferredoxin reductases revealed the presence of conserved sequence motifs in the FAD-binding domain as well as several highly conserved residues involved in FAD and NAD cofactor binding. Moreover, the NAD-binding site of FdR9 contains a modified Rossmann-fold motif, GxSxxS, instead of the classical GxGxxG motif.

Substrate Channeling via a Transient Protein-Protein Complex: The case of D-Glyceraldehyde-3-Phosphate Dehydrogenase and L-Lactate Dehydrogenase

Substrate channeling studies have frequently failed to provide conclusive results due to poor understanding of this subtle phenomenon. We analyzed the mechanism of NADH-channeling from D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to L-lactate Dehydrogenase (LDH) using enzymes from different cells. Enzyme kinetics studies showed that LDH activity with free NADH and GAPDH-NADH complex always take place in parallel. The channeling is observed only in assays that mimic cytosolic conditions where free NADH concentration is negligible and the GAPDH-NADH complex is dominant. Molecular dynamics and protein-protein interaction studies showed that LDH and GAPDH can form a leaky channeling complex only at the limiting NADH concentrations. Surface calculations showed that positive electric field between the NAD(H) binding sites on LDH and GAPDH tetramers can merge in the LDH-GAPDH complex. NAD(H)-channeling within the LDH-GAPDH complex can be an extension of NAD(H)-channeling within each tetramer. In the case of a transient LDH-(GAPDH-NADH) complex, the relative contribution from the channeled and the diffusive paths depends on the overlap between the off-rates for the LDH-(GAPDH-NADH) complex and the GAPDH-NADH complex. Molecular evolution or metabolic engineering protocols can exploit substrate channeling for metabolic flux control by fine-tuning substrate-binding affinity for the key enzymes in the competing reaction paths.

Structural and mutational analyses of the bifunctional arginine dihydrolase and ornithine cyclodeaminase AgrE from the cyanobacterium Anabaena

In cyanobacteria, metabolic pathways that use the nitrogen-rich amino acid arginine play a pivotal role in nitrogen storage and mobilization. The N-terminal domains of two recently identified bacterial enzymes: ArgZ from Synechocystis and AgrE from Anabaena, have been found to contain an arginine dihydrolase. This enzyme provides catabolic activity that converts arginine to ornithine, resulting in concomitant release of CO2 and ammonia. In Synechocystis, the ArgZ-mediated ornithine-ammonia cycle plays a central role in nitrogen storage and remobilization. The C-terminal domain of AgrE contains an ornithine cyclodeaminase responsible for the formation of proline from ornithine and ammonia production, indicating that AgrE is a bifunctional enzyme catalyzing two sequential reactions in arginine catabolism. Here, the crystal structures of AgrE in three different ligation states revealed that it has a tetrameric conformation, possesses a binding site for the arginine dihydrolase substrate l-arginine and product l-ornithine, and contains a binding site for the coenzyme NAD(H) required for ornithine cyclodeaminase activity. Structure-function analyses indicated that the structure and catalytic mechanism of arginine dihydrolase in AgrE are highly homologous with those of a known bacterial arginine hydrolase. We found that in addition to other active-site residues, Asn-71 is essential for AgrE's dihydrolase activity. Further analysis suggested the presence of a passage for substrate channeling between the two distinct AgrE active sites, which are situated ∼45 Å apart. These results provide structural and functional insights into the bifunctional arginine dihydrolase-ornithine cyclodeaminase enzyme AgrE required for arginine catabolism in Anabaena.

Antioxidative Pseudoenzymatic Mechanism of NAD(P)H Coexisting with Oxyhemoglobin for Suppressed Methemoglobin Formation.

Oxyhemoglobin (HbO2) coexisting with equimolar NADH retards autoxidation and oxidant-induced metHb formation based on the pseudocatalase (CAT) and pseudosuperoxide dismutase (SOD) activities. In this work, we compared the effects of NADH with those of NADPH and estimated the binding site of NAD(P)H to HbO2 to elucidate the antioxidative mechanisms. The results clarified that pseudo-CAT and pseudo-SOD activities of HbO2 coexisting with NADPH were similar to activities obtained with NADH. Prompt MetHb formation (<40 min) facilitated by oxidants (H2O2, NO, and NaNO2) was hindered by NADPH. These effects were similar to those of NADH. However, we found that NADPH is thermally unstable compared to NADH and that NADPH cannot sustain antioxidative effects for a long period of autoxidation to metHb such as 24 h. Lineweaver-Burk plots clarified that the Michaelis constants of these pseudoenzymatic activities are in the millimolar range. Addition of inositol hexaphosphate (IHP) and 2,3-diphosphoglycerate (DPG), which are known to bind not only with deoxyHb but also weakly with HbO2, showed competitive inhibition of pseudoenzymatic activities. These results suggest that the binding site of NADH and NADPH on HbO2 is the same as those of IHP and DPG. 31P nuclear magnetic resonance definitively showed 1:1 stoichiometric binding of NADH to HbO2. High-performance liquid chromatography analysis showed that NADH preferentially inhibited autoxidation of α-subunit heme. Docking simulations also predicted that the binding site of relaxed-state HbO2 with NAD(P)H is the same as those with IHP and DPG. Collectively, the pseudoenzymatic activities of HbO2 coexisting with NAD(P)H are induced by the 1:1 stoichiometric binding of NAD(P)H to HbO2.

Gene Cloning and Gene Expression Characteristics of Alcohol Dehydrogenase in Osmanthus fragrans var. semperflorens

Because of a lot of aroma components in the flowers, Osmanthus fragrans is widely used as ornamental tree species and its flowers is used as materials of spices and food additives. Alcohol dehydrogenase (ADH) plays an important regulation role in growth, development, stress resistance and aroma synthesis of plants. Using the flowers of O fragrans var. semperflorens as materials, the full-length cDNA of ADH gene (termed OfADH) was successfully cloned through rapid amplification of cDNA ends (RACE), which has the 1128 bp open reading frame (ORF) in length and codes 375 amino acids. The results of conserved domain analysis show that the OfADH belongs to the zinc-dependent medium-chain dehydrogenase /reductase (MDR) superfamily, contains 22 NAD(P) binding sites in the liver_alcohol_DH_like conserved domains, and also has the PLN02740 (Alcohol dehydrogenase-like) domain. The results of semi-quantitative RT-PCR indicate that the expression level of OfADH in O. fragrans flowers is consistent with the rule of aroma formation and release, while the expression of OfADH in leaves is positively correlated with high temperature stress, suggesting that OfADH has obvious functional diversity in regulation of aroma synthesis and response to high temperature stress.

Systematic analysis and integrative discovery of active-site subpocket-specific dehydroquinate synthase inhibitors combating antibiotic-resistant Staphylococcus aureus infection.

Shikimate pathway plays an essential role in the biosynthesis of aromatic amino acids in various plants and bacteria, which consists of seven key enzymes and they are all attractive targets for antibacterial agent development due to their absence in humans. The Staphylococcus aureus dehydroquinate synthase (SaDHQS)is involved in the second step of shikimate pathway, which catalyzes the NAD <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:msup><mml:mrow/><mml:mrow><mml:mo>+</mml:mo></mml:mrow></mml:msup></mml:math> -dependent conversion of 3-deoxy-D-arabino-heptulosonate-7-phosphate to dehydroquinate via multiple steps. The enzyme active site can be characterized by two spatially separated subpockets 1 and 2, which represent the reaction center of substrate adduct with NAD <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:msup><mml:mrow/><mml:mrow><mml:mo>+</mml:mo></mml:mrow></mml:msup></mml:math> nicotinamide moiety and the assistant binding site of NAD <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:msup><mml:mrow/><mml:mrow><mml:mo>+</mml:mo></mml:mrow></mml:msup></mml:math> adenine moiety, respectively. In silico virtual screening is performed against a biogenic compound library to discover SaDHQS subpocket-specific inhibitors, which were then tested against both antibiotic-sensitive and antibiotic-resistant S. aureus strains by using in vitro susceptibility test. The activity profile of hit compounds has no considerable difference between the antibiotic-sensitive and -resistant strains. The subpocket 1-specific inhibitors exhibit a generally higher activity than subpocket 2-specific inhibitors, and they also hold a strong selectivity between their cognate and noncognate subpockets. Dynamics and energetics analyses reveal that the SaDHQS active site prefers to interact with amphipathic and polar inhibitors by forming multiple hydrogen bonds and van der Waals packing at the complex interfaces of the two subpockets with their cognate inhibitors.

Substitutions of a buried glutamate residue hinder the conformational change in horse liver alcohol dehydrogenase and yield a surprising complex with endogenous 3′-Dephosphocoenzyme A

Glu-267 is highly conserved in alcohol dehydrogenases and buried as a negatively-charged residue in a loop of the NAD coenzyme binding domain. Glu-267 might have a structural role and contribute to a rate-promoting vibration that facilitates catalysis. Substitutions of Glu-267 with histidine or asparagine residues increase the dissociation constants for the coenzymes (NAD+ by ∼40-fold, NADH by ∼200-fold) and significantly decrease catalytic efficiencies by 16–1200-fold various substrates and substituted enzymes. The turnover numbers modestly change with the substitutions, but hydride transfer is at least partially rate-limiting for turnover for alcohol oxidation. X-ray structures of the E267H and E267 N enzymes are similar to the apoenzyme (open) conformation of the wild-type enzyme, and the substitutions are accommodated by local changes in the structure. Surprisingly, the E267H and E267 N enzymes have endogenous (from the expression in E. coli) 3′-dephosphocoenzyme A bound in the active site with the ADP moiety in the NAD binding site and the pantethiene sulfhydryl bound to the catalytic zinc. The kinetics and crystallography show that the substitutions of Glu-267 hinder the conformational change, which occurs when wild-type enzyme binds coenzymes, and affect productive binding of substrates.

Effects of hydroxycinnamic acids on the glycolysis pathway

Glycolysis is a metabolic pathway vital to the production of energy and some organisms rely on it solely to meet their energy requirements. It is also a central pathway in the metabolism of carbohydrates and a source of therapeutic targets against diabetes and cancer. Caffeoylquinic acids (CQAs) have been extensively studied for their role in the treatment and prevention of diabetes (and cancer) but their mechanisms of action remain mostly unknown. As such, molecular docking was used to find possible targets of CQAs in the glycolysis pathway. The molecular docking assays showed that CQAs were docked preferably to the Rossman fold (nicotinamide adenine dinucleotide — NAD(H) binding site) of oxidoreductases, that use NAD(H) as a cofactor, than to any other site. In-vitro assays were then performed using two NAD(H) dependent oxidoreductases from glycolysis (alcohol dehydrogenase and L-lactate dehydrogenase) in order confirm if CQAs would compete with the cofactor to inhibit the reaction. The results from these assays indicate that CQAs can act as both inhibitors and activators of NAD(H) dependent oxidoreductases of the glycolysis pathway.

Insights into the inhibitory mechanisms of NADH on the \u03b1\u03b3 heterodimer of human NAD-dependent isocitrate dehydrogenase

Human NAD-dependent isocitrate dehydrogenase (NAD-IDH) catalyzes the oxidative decarboxylation of isocitrate in the citric acid cycle. In the α2βγ heterotetramer of NAD-IDH, the γ subunit plays the regulatory role and the β subunit the structural role. Previous biochemical data have shown that mammalian NAD-IDHs can be inhibited by NADH; however, the molecular mechanism is unclear. In this work, we show that the αβ, αγ and α2βγ enzymes of human NAD-IDH can be inhibited by NADH, and further determine the crystal structure of the αγ heterodimer bound with an Mg2+ and an NADH at the active site and an NADH at the allosteric site, which resembles that of the inactive αMgγ heterodimer. The NADH at the active site occupies the binding site for NAD+ and prevents the binding of the cofactor. The NADH at the allosteric site occupies the binding sites for ADP and citrate and blocks the binding of the activators. The biochemical data confirm that the NADH binding competes with the binding of NAD+ and the binding of citrate and ADP, and the two effects together contribute to the NADH inhibition on the activity. These findings provide insights into the inhibitory mechanisms of the αγ heterodimer by NADH.

Abstract 5241: Using affinity probes to explore the nephrotoxicity of aristolochic acid

Abstract Aristolochic acid, AA, is a natural compound found in Aristolochia plants used in Chinese Traditional Medicine and is a component of many herbal remedies used worldwide. In humans, exposure to AA is associated with kidney toxicity and otherwise rare upper urinary tract cancer (UTUC). AA undergoes bioactivation creating reactive species of AA that bind covalently to proteins and DNA. Formation of mutagenic AA-DNA adducts leads to AA-induced cancers. We identified the mutational signature associated with AA exposure in patients with UTUC in Taiwan, as well as in AA-initiated cancers in liver, bladder and kidneys. Meanwhile, the mechanism of AA nephrotoxicity remained a mystery, with covalent and non-covalent binding of AA or its metabolites to proteins hypothesized to be involved in its nephrotoxic effects. As millions of people in Asia have ingested AA and no effective therapies are known, establishing the molecular mechanism(s) of AA nephrotoxicity is a topic of major importance. A series of AA analogs were used to design affinity probes for target identification. Structure activity relationship studies in human cells aided in identifying a position in AA suitable for introducing biotinylated, aminopropyl and alkyne linkers. In addition, we have developed and validated monoclonal antibodies against AA-bovine serum albumin and/or AA-DNA covalent adducts. Combined with proteomics techniques, AA-affinity probes and monoclonal antibodies provide powerful tools for defining AA targets in human kidneys. We attached the aminopropyloxy derivative of AA to magnetic beads coated with N-hydroxysuccinimido acyl linkers. These probes, in parallel with unmodified, “control” beads were incubated with renal cortex lysates obtained from C3H mice selected for their sensitivity to the effects of AA. Bound proteins were digested "on bead", then identified by mass spectroscopic peptide analysis, with a total of 316 bound proteins detected. Spectral counts for 128 proteins bound to AA-beads were higher than those for control beads. After correction for nonspecific binding, 18 of these proteins had spectral counts of 10 or higher. These proteins are localized to various organelles, indicating high coverage of the major subcellular compartments. Among these proteins, Cryzl2, quinone oxidoreductase-like 2, was the leading candidate. Based on homology, Cryzl2 belongs to the family of the quinone oxidoreductase-like protein and bears a mitochondrial localization sequence. This protein retains conserved domains for both quinone oxidoreductase activity and NAD(P) binding sites, which is of particular interest since other oxidoreductases, such as NQO1, are known to catalyze nitroreduction, the initial step in the bioactivation of AA. In summary, our studies provide guidelines for design of AA-affinity probes and reveal an enzyme that may be responsible for AA activation in mitochondria, by generating active intermediates and inducing mitochondrial damage in renal tissue. Citation Format: Viktoriya S. Sidorenko, Kathleen G. Dickman, Thomas Rosenquist, Radha Bonala, Sivaprasad Attaluri, Irina Zaitseva, Charles Iden, Francis Johnson, Arthur P. Grollman. Using affinity probes to explore the nephrotoxicity of aristolochic acid [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5241. doi:10.1158/1538-7445.AM2017-5241

Mycofactocin-associated mycobacterial dehydrogenases with non-exchangeable NAD cofactors

During human infection, Mycobacterium tuberculosis (Mtb) survives the normally bacteriocidal phagosome of macrophages. Mtb and related species may be able to combat this harsh acidic environment which contains reactive oxygen species due to the mycobacterial genomes encoding a large number of dehydrogenases. Typically, dehydrogenase cofactor binding sites are open to solvent, which allows NAD/NADH exchange to support multiple turnover. Interestingly, mycobacterial short chain dehydrogenases/reductases (SDRs) within family TIGR03971 contain an insertion at the NAD binding site. Here we present crystal structures of 9 mycobacterial SDRs in which the insertion buries the NAD cofactor except for a small portion of the nicotinamide ring. Line broadening and STD-NMR experiments did not show NAD or NADH exchange on the NMR timescale. STD-NMR demonstrated binding of the potential substrate carveol, the potential product carvone, the inhibitor tricyclazol, and an external redox partner 2,6-dichloroindophenol (DCIP). Therefore, these SDRs appear to contain a non-exchangeable NAD cofactor and may rely on an external redox partner, rather than cofactor exchange, for multiple turnover. Incidentally, these genes always appear in conjunction with the mftA gene, which encodes the short peptide MftA, and with other genes proposed to convert MftA into the external redox partner mycofactocin.

Three-Dimensional Model of Human Nicotinamide Nucleotide Transhydrogenase (NNT) and Sequence-Structure Analysis of its Disease-Causing Variations.

ABSTRACTDefective mitochondrial proteins are emerging as major contributors to human disease. Nicotinamide nucleotide transhydrogenase (NNT), a widely expressed mitochondrial protein, has a crucial role in the defence against oxidative stress. NNT variations have recently been reported in patients with familial glucocorticoid deficiency (FGD) and in patients with heart failure. Moreover, knockout animal models suggest that NNT has a major role in diabetes mellitus and obesity. In this study, we used experimental structures of bacterial transhydrogenases to generate a structural model of human NNT (H‐NNT). Structure‐based analysis allowed the identification of H‐NNT residues forming the NAD binding site, the proton canal and the large interaction site on the H‐NNT dimer. In addition, we were able to identify key motifs that allow conformational changes adopted by domain III in relation to its functional status, such as the flexible linker between domains II and III and the salt bridge formed by H‐NNT Arg882 and Asp830. Moreover, integration of sequence and structure data allowed us to study the structural and functional effect of deleterious amino acid substitutions causing FGD and left ventricular non‐compaction cardiomyopathy. In conclusion, interpretation of the function–structure relationship of H‐NNT contributes to our understanding of mitochondrial disorders.

Crystal structure of archaeal ketopantoate reductase complexed with coenzyme a and 2-oxopantoate provides structural insights into feedback regulation.

Coenzyme A (CoA) plays essential roles in a variety of metabolic pathways in all three domains of life. The biosynthesis pathway of CoA is strictly regulated by feedback inhibition. In bacteria and eukaryotes, pantothenate kinase is the target of feedback inhibition by CoA. Recent biochemical studies have identified ketopantoate reductase (KPR), which catalyzes the NAD(P)H-dependent reduction of 2-oxopantoate to pantoate, as a target of the feedback inhibition by CoA in archaea. However, the mechanism for recognition of CoA by KPR is still unknown. Here we report the crystal structure of KPR from Thermococcus kodakarensis in complex with CoA and 2-oxopantoate. CoA occupies the binding site of NAD(P)H, explaining the competitive inhibition by CoA. Our structure reveals a disulfide bond between CoA and Cys84 that indicates an irreversible inhibition upon binding of CoA. The structure also suggests the cooperative binding of CoA and 2-oxopantoate that triggers a conformational closure and seems to facilitate the disulfide bond formation. Our findings provide novel insights into the mechanism that regulates biosynthesis of CoA in archaea.

SIRT5 Mutants Reveal Insights into Sirtuin Regulation

Sirtuins are emerging as key regulators for metabolism and aging. These regulations were primarily attributed to sirtuin's NAD+ dependent deacetylase activity. Among the seven human sirtuins, SIRT5 is the only one that also catalyzes desuccinylation. It is becoming clear that sirtuins have evolved to adapt different regulatory mechanisms of their activities. However, detailed studies on substrate specificity, isozyme selectivity, and inhibition mechanisms of sirtuins are still lacking. Two amino acids—Y102 and R105—are responsible for SIRT5′s desuccinylation activity. Therefore, we made single and double muatations on the two residues and compared their steady‐state kinetic features with those of wild type SIRT5 and SIRT1. We noticed that Y102 and R105 account partially for the differential substrate specificity and drug isoform selectivity of SIRT5, implying that sirtuin activity is also regulated by the dynamic flexibility in the protein structures. Our inhibition analysis showed that nicotinamide inhibited SIRT5′s desuccinylation activity by competing with NAD+; while it inhibited SIRT1′s and SIRT5′s deacetylation activity via a combination of two pathways—competing with NAD+ and base‐exchange. Combining it with our bisubstrate kinetic analysis, we conclude that the binding of succinylated substrate creates a tighter binding site for both NAD+ and nicotinamide, generating alternative nicotinamide regulations of SIRT5. Our research studies provide insight into Sirtuin regulation andthe rationale designing of sirtuin isoform selective inhibitors.