The conserved glycine/alanine residue of the active-site loop containing the putative acetylCoA-binding motif is essential for the overall structural integrity of Mesorhizobium loti arylamine N-acetyltransferase 1

The arylamine N-acetyltransferases are important xenobiotic-metabolizing enzymes that catalyze an acetyl group transfer from acetylCoA to arylamine substrates. NAT enzymes possess an active-site loop (the active-site P-loop) involved in substrate binding and selectivity. The Gly/Ala residue present at the start of the active-site P-loop, although conserved in all NAT enzymes, is not involved in the catalytic mechanism or substrate binding. Here we show that a small amino acid (such as Gly or Ala) at this position is important not only for maintaining the functions of the active-site P-loop but, more surprisingly, also important for maintaining the overall structural integrity of NAT enzymes. Our data thus suggest that in addition to its role in substrate binding and selectivity, the active-site P-loop could play a wider structural role in NAT enzymes.     

Sphingosine kinase: Role in regulation of bioactive sphingolipid mediators in inflammation

Sphingolipids and their synthetic enzymes are emerging as important mediators in inflammatory responses and as regulators of immune cell functions. In particular, sphingosine kinase (SK) and its product sphingosine-1-phosphate (S1P) have been extensively implicated in these processes. SK catalyzes the phosphorylation of sphingosine to S1P and exists as two isoforms, SK1 and SK2. SK1 has been shown to be activated by cytokines including tumor necrosis factor-alpha (TNF-α) and interleukin1-β (IL1-β). The activation of SK1 in this pathway has been shown to be, at least in part, required for mediating TNF-α and IL1-β inflammatory responses in cells, including induction of cyclo-oxygenase 2 (COX2). In addition to their role in inflammatory signaling, SK and S1P have also been implicated in various immune cell functions including, mast cell degranulation, migration of neutrophils, and migration and maturation of lymphocytes. The involvement of sphingolipids and sphingolipid metabolizing enzymes in inflammatory signaling and immune cell functions has implicated these mediators in numerous inflammatory disease states as well. The contribution of these mediators, specifically SK1 and S1P, to inflammation and disease are discussed in this review.     

Substrate proton to heme distances in CYP2C9 allelic variants and alterations by the heterotropic activator, dapsone

CYP2C9 polymorphisms result in reduced enzyme catalytic activity and greater activation by effector molecules as compared to wild-type protein, with the mechanism(s) for these changes in activity not fully elucidated. Through T₁ NMR and spectral binding analyses, mechanism(s) for these differences in behavior of the variant proteins (CYP2C9.2, CYP2C9.3, and CYP2C9.5) as compared to CYP2C9.1 were assessed. Neither altered binding affinity nor substrate (flurbiprofen) proton to heme-iron distances differed substantially among the four enzymes. Co-incubation with dapsone resulted in reduced substrate proton to heme-iron distances for all enzymes, providing at least a partial mechanism for the activation of CYP2C9 variants by dapsone. In summary, neither altered binding affinity nor substrate orientation appear to be major factors in the reduced catalytic activity noted in the CYP2C9 variants, but dapsone co-incubation caused similar changes in substrate proton to heme-iron distances suggesting at least partial common mechanisms in the activation of the CYP2C9 forms.     

Substrate kringle-mediated catalysis by the streptokinase-plasmin activator complex: Critical contribution of kringle-4 revealed by the mutagenesis approaches

Streptokinase (SK) is a protein co-factor with a potent capability for human plasminogen (HPG) activation. Our previous studies [1] have indicated a major role of long-range protein-protein contacts between the three domains (alpha, beta, and gamma) of SK and the multi-domain HPG substrate (K1-K5CD). To further explore this phenomenon, we prepared truncated derivatives of HPG with progressive removal of kringle domains, like K5CD, K4K5CD, K3-K5CD (K3K4K5CD), K2-K5CD (K2K3K4K5CD) and K1-K5CD (K1K2K3K4K5CD). While urokinase (uPA) cleaved the scissile peptide in the isolated catalytic domain (μPG) with nearly the same rate as with full-length HPG, SK-plasmin showed only 1-2% activity, revealing mutually distinct mechanisms of HPG catalysis between the eukaryotic and prokaryotic activators. Remarkably, with SK.HPN (plasmin), the ‘addition’ of both kringles 4 and 5 onto the catalytic domain showed catalytic rates comparable to full length HPG, thus identifying the dependency of the “long-range” enzyme-substrate interactions onto these two CD-proximal domains. Further, chimeric variants of K5CD were generated by swapping the kringle domains of HPG with those of uPA and TPA (tissue plasminogen activator), separately. Surprisingly, although native-like catalytic turnover rates were retained when either K1, K2 or K4 of HPG was substituted at the K5 position in K5CD, these were invariably lost once substituted with the evolutionarily more distant TPA- and uPA-derived kringles. The present results unveil a novel mechanism of SK.HPN action in which augmented catalysis occurs through enzyme-substrate interactions centered on regions in substrate HPG (kringles 4 and 5) that are spatially distant from the scissile peptide bond.     

Structure and function of the transketolase from Mycobacterium tuberculosis and comparison with the human enzyme

The transketolase (TKT) enzyme in Mycobacterium tuberculosis represents a novel drug target for tuberculosis treatment and has low homology with the orthologous human enzyme. Here, we report on the structural and kinetic characterization of the transketolase from M. tuberculosis (TBTKT), a homodimer whose monomers each comprise 700 amino acids. We show that TBTKT catalyses the oxidation of donor sugars xylulose-5-phosphate and fructose-6-phosphate as well as the reduction of the acceptor sugar ribose-5-phosphate. An invariant residue of the TKT consensus sequence required for thiamine cofactor binding is mutated in TBTKT; yet its catalytic activities are unaffected, and the 2.5 Å resolution structure of full-length TBTKT provides an explanation for this. Key structural differences between the human and mycobacterial TKT enzymes that impact both substrate and cofactor recognition and binding were uncovered. These changes explain the kinetic differences between TBTKT and its human counterpart, and their differential inhibition by small molecules. The availability of a detailed structural model of TBTKT will enable differences between human and M. tuberculosis TKT structures to be exploited to design selective inhibitors with potential antitubercular activity.     

The role of Y84 on domain 1 and Y87 on domain 2 of Paragonimus westermani taurocyamine kinase: Insights on the substrate binding mechanism of a trematode phosphagen kinase

The two-domain taurocyamine kinase (TK) from Paragonimus westermani was suggested to have a unique substrate binding mechanism. We performed site-directed mutagenesis on each domain of this TK and compared the kinetic parameters Km^Tc and Vmax with that of the wild-type to determine putative amino acids involved in substrate recognition and binding. Replacement of Y84 on domain 1 and Y87 on domain 2 with R resulted in the loss of activity for the substrate taurocyamine. Y84E mutant has a dramatic decrease in affinity and activity for taurocyamine while Y87E has completely lost catalytic activity. Substituting H and I on the said positions also resulted in significant changes in activity. Mutation of the residues A59 on the GS region of domain 1 also caused significant decrease in affinity and activity while mutation on the equivalent position on domain 2 resulted in complete loss of activity.     

S-(4-Nitrophenacyl)glutathione is a specific substrate for glutathione transferase omega 1-1

Glutathione transferase omega 1-1 (GSTO1-1) catalyzes the biotransformation of arsenic and is implicated as a factor influencing the age-at-onset of Alzheimer’s disease and the posttranslational activation of interleukin 1β (IL-1β). Investigation of the biological role of GSTO1-1 variants has been hampered by the lack of a specific assay for GSTO1-1 activity in tissue samples that contain other GSTs and other enzymes with similar catalytic specificities. Previous studies (P. G. Board and M. W. Anders, Chem. Res. Toxicol. 20 (2007) 149-154) have shown that GSTO1-1 catalyzes the reduction of S-(phenacyl)glutathiones to acetophenones. A new substrate, S-(4-nitrophenacyl)glutathione (4NPG), has been prepared and found to have a high turnover with GSTO1-1 but negligible activity with GSTO2-2 and other members of the glutathione transferase superfamily. A spectrophotometric assay with 4NPG as a substrate has been used to determine GSTO1-1 activity in several human breast cancer cell lines and in mouse liver and brain tissues.     

An assay for sphingosine kinase activity using biotinylated sphingosine and streptavidin-coated membranes

Sphingosine kinase catalyses the phosphorylation of sphingosine to generate sphingosine 1-phosphate, a lipid signaling molecule implicated in roles in a diverse range of mammalian cell processes through its action as both a ligand for G-protein-coupled cell-surface receptors and an apparent intracellular second messenger. This paper describes a rapid, sensitive, and reproducible assay for sphingosine kinase activity using biotinylated sphingosine (biotinyl-Sph) as a substrate and capturing the phosphorylated product with streptavidin-coated membranes. We have shown that both human sphingosine kinase 1 and 2 (hSK1 and hSK2) can efficiently phosphorylate biotinyl-Sph, with K(m) values similar to those of sphingosine. The assay utilizing this substrate has high sensitivity for hSK1 and hSK2, with detection limits in the low-femtomole range for both purified recombinant enzymes. Importantly, we have also demonstrated the capacity of this assay to measure endogenous sphingosine kinase activity in crude cell extracts and to follow changes in this activity following sphingosine kinase activation. Together, these results demonstrate the potential utility of this assay in both cell-based analysis of sphingosine kinase signaling pathways and high-throughput screens for agents affecting sphingosine kinase activity in vitro.     

Crystal structure of isoflavone reductase from alfalfa (Medicago sativa L.)

Isoflavonoids play important roles in plant defense and exhibit a range of mammalian health-promoting activities. Isoflavone reductase (IFR) specifically recognizes isoflavones and catalyzes a stereospecific NADPH-dependent reduction to (3R)-isoflavanone. The crystal structure of Medicago sativa IFR with deletion of residues 39-47 has been determined at 1.6A resolution. Structural analysis, molecular modeling and docking, and comparison with the structures of other NADPH-dependent enzymes, defined the putative binding sites for co-factor and substrate and potential key residues for enzyme activity and substrate specificity. Further mutagenesis has confirmed the role of Lys144 as a catalytic residue. This study provides a structural basis for understanding the enzymatic mechanism and substrate specificity of IFRs as well as the functions of IFR-like proteins.     

Eukaryotic elongation factor 1A interacts with sphingosine kinase and directly enhances its catalytic activity

Sphingosine 1-phosphate (S1P) has many important roles in mammalian cells, including contributing to the control of cell survival and proliferation. S1P is generated by sphingosine kinases (SKs), of which two mammalian isoforms have been identified (SK1 and SK2). To gain a better understanding of SK regulation, we have used a yeast two-hybrid screen to identify SK1-interacting proteins and established elongation factor 1A (eEF1A) as one such protein that associates with both SK1 and SK2. We show the direct interaction of eEF1A with the SKs in vitro, whereas the physiological relevance of this association was demonstrated by co-immunoprecipitation of the endogenous proteins from cell lysates. Although the canonical role of eEF1A resides in protein synthesis, it has also been implicated in other roles, including regulating the activity of some signaling enzymes. Thus, we examined the potential role of eEF1A in regulation of the SKs and show that eEF1A is able to directly increase the activity of SK1 and SK2 approximately 3-fold in vitro. Substrate kinetics demonstrated that eEF1A increased the catalytic rate of both SKs, while having no observable effect on substrate affinities of these enzymes for either ATP or sphingosine. Overexpression of eEF1A in quiescent Chinese hamster ovary cells increased cellular SK activity, whereas a small interfering RNA-mediated decrease in eEF1A levels in MCF7 cells substantially reduced cellular SK activity and S1P levels, supporting the in vivo physiological relevance of this interaction. Thus, this study has established a novel mechanism of regulation of both SK1 and SK2 that is mediated by their interaction with eEF1A.     

The compartmentalization and translocation of the sphingosine kinases: Mechanisms and functions in cell signaling and sphingolipid metabolism

Members of the sphingosine kinase (SK) family of lipid signaling enzymes, comprising SK1 and SK2 in humans, are receiving considerable attention for their roles in a number of physiological and pathophysiological processes. The SKs are considered signaling enzymes based on their production of the potent lipid second messenger sphingosine-1-phosphate, which is the ligand for a family of five G-protein-linked receptors. Both SK1 and SK2 are intracellular enzymes and do not possess obvious membrane anchor domains within their primary sequences. The native substrates (sphingosine and dihydrosphingosine) are lipids, as are the corresponding products, and therefore would have a propensity to be membrane associated, suggesting that specific membrane localization of the SKs could affect both access to substrate and localized production of product. Here, we consider the emerging picture of the SKs as enzymes localized to specific intracellular sites, sometimes by agonist-dependent translocation, the mechanism targeting these enzymes to those sites, and the functional consequence of that localization. Not only is the signaling output of the SKs affected by subcellular localization, but the role of these enzymes as metabolic regulators of sphingolipid metabolism may be impacted as well.     

Biochemical and structural characterization of polyphosphate kinase 2 from the intracellular pathogen Francisella tularensis

The metabolism of polyphosphate is important for the virulence of a wide range of pathogenic bacteria and the enzymes of polyphosphate metabolism have been proposed as an anti-bacterial target. In the intracellular pathogen Francisella tularensis, the product of the gene FTT1564 has been identified as a polyphosphate kinase from the polyphosphate kinase 2 (PPK2) family. The isogenic deletion mutant was defective for intracellular growth in macrophages and was attenuated in mice, indicating an important role for polyphosphate in the virulence of Francisella. Herein, we report the biochemical and structural characterization of F. tularensis polyphosphate kinase (FtPPK2) with a view to characterizing the enzyme as a novel target for inhibitors. Using an HPLC-based activity assay, the substrate specificity of FtPPK2 was found to include purine but not pyrimidine nts. The activity was also measured using ³¹P-NMR. FtPPK2 has been crystallized and the structure determined to 2.23 Å (1 Å=0.1 nm) resolution. The structure consists of a six-stranded parallel β-sheet surrounded by 12 α-helices, with a high degree of similarity to other members of the PPK2 family and the thymidylate kinase superfamily. Residues proposed to be important for substrate binding and catalysis have been identified in the structure, including a lid-loop and the conserved Walker A and B motifs. The ΔFTT1564 strain showed significantly increased sensitivity to a range of antibiotics in a manner independent of the mode of action of the antibiotic. This combination of biochemical, structural and microbiological data provide a sound foundation for future studies targeting the development of PPK2 small molecule inhibitors.     

Lipid phosphate phosphatase (LPP3) and vascular development

Lipid phosphate phosphatases (LPP) are integral membrane proteins with broad substrate specificity that dephosphorylate lipid substrates including phosphatidic acid, lysophosphatidic acid, ceramide 1-phosphate, sphingosine 1-phosphate, and diacylglycerol pyrophosphate. Although the three mammalian enzymes (LPP1-3) demonstrate overlapping catalytic activities and substrate preferences in vitro, the phenotypes of mice with targeted inactivation of the Ppap2 genes encoding the LPP enzymes reveal nonredundant functions. A specific role for LPP3 in vascular development has emerged from studies of mice lacking Ppap2b. A meta-analysis of multiple, large genome-wide association studies identified a single nucleotide polymorphism in PPAP2B as a novel predictor of coronary artery disease. In this review, we will discuss the evidence that links LPP3 to vascular development and disease and evaluate potential molecular mechanisms. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

Epsilon amino caproic acid inhibits streptokinase-plasminogen activator complex formation and substrate binding through kringle-dependent mechanisms

Lysine side chains induce conformational changes in plasminogen (Pg) that regulate the process of fibrinolysis or blood clot dissolution. A lysine side-chain mimic, epsilon amino caproic acid (EACA), enhances the activation of Pg by urinary-type and tissue-type Pg activators but inhibits Pg activation induced by streptokinase (SK). Our studies of the mechanism of this inhibition revealed that EACA (IC(50) 10 microM) also potently blocked amidolytic activity by SK and Pg at doses nearly 10000-fold lower than that required to inhibit the amidolytic activity of plasmin. Different Pg fragments were used to assess the role of the kringles in mediating the inhibitory effects of EACA: mini-Pg which lacks kringles 1-4 of Glu-Pg and micro-Pg which lacks all kringles and contains only the catalytic domain. SK bound with similar affinities to Glu-Pg (K(A) = 2.3 x 10(9) M(-1)) and to mini-Pg (K(A) = 3.8 x 10(9) M(-)(1)) but with significantly lower affinity to micro-Pg (K(A) = 6 x 10(7) M(-)(1)). EACA potently inhibited the binding of Glu-Pg to SK (K(i) = 5.7 microM), but was less potent (K(i) = 81.1 microM) for inhibiting the binding of mini-Pg to SK and had no significant inhibitory effects on the binding of micro-Pg and SK. In assays simulating substrate binding, EACA also potently inhibited the binding of Glu-Pg to the SK-Glu-Pg activator complex, but had negligible effects on micro-Pg binding. Taken together, these studies indicate that EACA inhibits Pg activation by blocking activator complex formation and substrate binding, through a kringle-dependent mechanism. Thus, in addition to interactions between SK and the protease domain, interactions between SK and the kringle domain(s) play a key role in Pg activation.     

The C-terminal end of the Trypanosoma brucei editing deaminase plays a critical role in tRNA binding

Adenosine to inosine editing at the wobble position allows decoding of multiple codons by a single tRNA. This reaction is catalyzed by adenosine deaminases acting on tRNA (ADATs) and is essential for viability. In bacteria, the anticodon-specific enzyme is a homodimer that recognizes a single tRNA substrate (tRNA(Arg)(ACG)) and can efficiently deaminate short anticodon stem-loop mimics of this tRNA in vitro. The eukaryal enzyme is composed of two nonidentical subunits, ADAT2 and ADAT3, which upon heterodimerization, recognize seven to eight different tRNAs as substrates, depending on the organism, and require a full-length tRNA for activity. Although crystallographic data have provided clues to why the bacterial deaminase can utilize short substrates, residues that provide substrate binding and recognition with the eukaryotic enzymes are not currently known. In the present study, we have used a combination of mutagenesis, binding studies, and kinetic analysis to explore the contribution of individual residues in Trypanosoma brucei ADAT2 (TbADAT2) to tRNA recognition. We show that deletion of the last 10 amino acids at the C terminus of TbADAT2 abolishes tRNA binding. In addition, single alanine replacements of a string of positively charged amino acids (KRKRK) lead to binding defects that correlate with losses in enzyme activity. This region, which we have termed the KR-domain, provides a first glance at key residues involved in tRNA binding by eukaryotic tRNA editing deaminases.     

A real-time high-throughput fluorescence assay for sphingosine kinases

Sphingosine kinases (SphKs), of which there are two isoforms, SphK1 and SphK2, have been implicated in regulation of many important cellular processes. We have developed an assay for monitoring SphK1 and SphK2 activity in real time without the need for organic partitioning of products, radioactive materials, or specialized equipment. The assay conveniently follows SphK-dependent changes in 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled sphingosine (Sph) fluorescence and can be easily performed in 384-well plate format with small reaction volumes. We present data showing dose-proportional responses to enzyme, substrate, and inhibitor concentrations. The SphK1 and SphK2 binding affinities for NBD-Sph and the IC₅₀ values of inhibitors determined were consistent with those reported with other methods. Because of the versatility and simplicity of the assay, it should facilitate the routine characterization of inhibitors and SphK mutants and can be readily used for compound library screening in high-throughput format.     

Structural and Biochemical Analysis of Human Pathogenic Astrovirus Serine Protease at 2.0 Å Resolution

Astroviruses are single-stranded RNA viruses with a replication strategy based on the proteolytic processing of a polyprotein precursor and subsequent release of the viral enzymes of replication. So far, the catalytic properties of the astrovirus protease as well as its structure have remained uncharacterized. In this study, the three-dimensional crystal structure of the predicted protease of human pathogenic astrovirus has been solved to 2.0 Å resolution. The protein displays the typical properties of trypsin-like enzymes but also several characteristic features: (i) a catalytic Asp-His-Ser triad in which the aspartate side chain is oriented away from the histidine, being replaced by a water molecule; (ii) a non-common conformation and composition of the S1 pocket; and (iii) the lack of the typical surface β-ribbons together with a “featureless” shape of the substrate-binding site. Hydrolytic activity assays indicate that the S1 pocket recognises Glu and Asp side chains specifically, which, therefore, are predicted to occupy the P1 position on the substrate cleavage site. The positive electrostatic potential featured by the S1 region underlies this specificity. The comparative structural analysis highlights the peculiarity of the astrovirus protease, and differentiates it from the human and viral serine proteases.     

Discovery of catalytically active orthologues of the Parkinson's disease kinase PINK1: analysis of substrate specificity and impact of mutations

Missense mutations of the phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) gene cause autosomal-recessive Parkinson's disease. To date, little is known about the intrinsic catalytic properties of PINK1 since the human enzyme displays such low kinase activity in vitro. We have discovered that, in contrast to mammalian PINK1, insect orthologues of PINK1 we have investigated-namely Drosophila melanogaster (dPINK1), Tribolium castaneum (TcPINK1) and Pediculus humanus corporis (PhcPINK1)-are active as judged by their ability to phosphorylate the generic substrate myelin basic protein. We have exploited the most active orthologue, TcPINK1, to assess its substrate specificity and elaborated a peptide substrate (PINKtide, KKWIpYRRSPRRR) that can be employed to quantify PINK1 kinase activity. Analysis of PINKtide variants reveal that PINK1 phosphorylates serine or threonine, but not tyrosine, and we show that PINK1 exhibits a preference for a proline at the +1 position relative to the phosphorylation site. We have also, for the first time, been able to investigate the effect of Parkinson's disease-associated PINK1 missense mutations, and found that nearly all those located within the kinase domain, as well as the C-terminal non-catalytic region, markedly suppress kinase activity. This emphasizes the crucial importance of PINK1 kinase activity in preventing the development of Parkinson's disease. Our findings will aid future studies aimed at understanding how the activity of PINK1 is regulated and the identification of physiological substrates.     

Intracellular localization of sphingosine kinase 1 alters access to substrate pools but does not affect the degradative fate of sphingosine-1-phosphate

Sphingosine kinase 1 (SK1) produces sphingosine-1-phosphate (S1P), a potent signaling lipid. The subcellular localization of SK1 can dictate its signaling function. Here, we use artificial targeting of SK1 to either the plasma membrane (PM) or the endoplasmic reticulum (ER) to test the effects of compartmentalization of SK1 on substrate utilization and downstream metabolism of S1P. Expression of untargeted or ER-targeted SK1, but surprisingly not PM-targeted SK1, results in a dramatic increase in the phosphorylation of dihydrosphingosine, a metabolic precursor in de novo ceramide synthesis. Conversely, knockdown of endogenous SK1 diminishes both dihydrosphingosine-1-phosphate and S1P levels. We tested the effects of SK1 localization on degradation of S1P by depletion of the ER-localized S1P phosphatases and lyase. Remarkably, S1P produced at the PM was degraded to the same extent as that produced in the ER. This indicates that there is an efficient mechanism for the transport of S1P from the PM to the ER. In acute labeling experiments, we find that S1P degradation is primarily driven by lyase cleavage of S1P. Counterintuitively, when S1P-specific phosphatases are depleted, acute labeling of S1P is significantly reduced, indicative of a phosphatase-dependent recycling process. We conclude that the localization of SK1 influences the substrate pools that it has access to and that S1P can rapidly translocate from the site where it is synthesized to other intracellular sites.51: 2546–2559.     

Beta sheet 2–alpha helix C loop of cytochrome P450 reductase serves as a docking site for redox partners

As a promiscuous redox partner, the biological role of cytochrome P450 reductase (CPR) depends significantly on protein–protein interactions. We tested a hypothesized CPR docking site by mutating D113, E115, and E116 to alanine and assaying activity toward various electron acceptors as a function of ionic strength. Steady-state cytochrome c studies demonstrated the mutations improved catalytic efficiency and decreased the impact of ionic strength on catalytic parameters when compared to wild type. Based on activity toward 7-ethoxy-4-trifluoro-methylcoumarin, CYP2B1 and CPR favored formation of an active CYP2B1•CPR complex and inactive (CYP2B1)₂•CPR complex until higher ionic strength whereby only the binary complex was observed. The mutations increased dissociation constants only for the binary complex and suppressed the ionic strength effect. Studies with a non-binding substrate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) suggest changes in activity toward cytochrome c and CYP2B1 reflect alterations in the route of electron transfer caused by the mutations. Electrostatic modeling of catalytic and binding parameters confirmed the importance of D113 and especially the double mutant E115 and E116 as mediators in forming charge–charge interactions between CPR and complex partners.