Substrate binding stabilizes S-adenosylhomocysteine hydrolase in a closed conformation

Comparison of crystal structures of S-adenosylhomocysteine (AdoHcy) hydrolase in the substrate-free, NAD(+) form [Hu, Y., Komoto, J., Huang, Y., Gomi, T., Ogawa, H., Takata, Y., Fujioka, M., and Takusagawa, F. (1999) Biochemistry 38, 8323-8333] and a substrate-bound, NADH form [Turner, M. A., Yuan, C.-S., Borchardt, R. T., Hershfield, M. S., Smith, G. D., and Howell, P. L. (1998) Nat. Struct. Biol. 5, 369-376] indicates large differences in the spatial arrangement of the catalytic and NAD(+) binding domains. The substrate-free, NAD(+) form exists in an "open" form with respect to catalytic and NAD(+) binding domains, whereas the substrate-bound, NADH form exists in a closed form with respect to those domains. To address whether domain closure is induced by substrate binding or its subsequent oxidation, we have measured the rotational dynamics of spectroscopic probes covalently bound to Cys(113) and Cys(421) within the catalytic and carboxyl-terminal domains. An independent domain motion is associated with the catalytic domain prior to substrate binding, suggesting the presence of a flexible hinge element between the catalytic and NAD(+) binding domains. Following binding of substrates (i.e., adenosine or neplanocin A) or a nonsubstrate (i.e., 3'-deoxyadenosine), the independent domain motion associated with the catalytic domain is essentially abolished. Likewise, there is a substantial decrease in the average hydrodynamic volume of the protein that is consistent with a reduction in the overall dimensions of the homotetrameric enzyme following substrate binding and oxidation observed in earlier crystallographic studies. Thus, the catalytic and NAD(+) binding domains are stabilized to form a closed active site through interactions with the substrate prior to substrate oxidation.     

PAR-1/MARK: a kinase essential for maintaining the dynamic state of microtubules

The serine/threonine kinase, PAR-1, is an essential component of the evolutionary-conserved polarity-regulating system, PAR-aPKC system, which plays indispensable roles in establishing asymmetric protein distributions and cell polarity in various biological contexts (Suzuki, A. and Ohno, S. (2006). J. Cell Sci., 119: 979-987; Matenia, D. and Mandelkow, E.M. (2009). Trends Biochem. Sci., 34: 332-342). PAR-1 is also known as MARK, which phosphorylates classical microtubule-associated proteins (MAPs) and detaches MAPs from microtubules (Matenia, D. and Mandelkow, E.M. (2009). Trends Biochem. Sci., 34: 332-342). This MARK activity of PAR-1 suggests its role in microtubule (MT) dynamics, but surprisingly, only few studies have been carried out to address this issue. Here, we summarize our recent study on live imaging analysis of MT dynamics in PAR-1b-depleted cells, which clearly demonstrated the positive role of PAR-1b in maintaining MT dynamics (Hayashi, K., Suzuki, A., Hirai, S., Kurihara, Y., Hoogenraad, C.C., and Ohno, S. (2011). J. Neurosci., 31: 12094-12103). Importantly, our results further revealed the novel physiological function of PAR-1b in maintaining dendritic spine morphology in mature neurons.     

The Formation of lysine tyrosylquinone (LTQ) is a self-processing reaction. Expression and characterization of a Drosophila lysyl oxidase

Recent work in our laboratory has established methods for the expression and purification of a recombinant form of Drosophila lysyl oxdidase (rDMLOXL-1) [Molnar, J., Ujfaludi, Z., Fong, S. F. T., Bollinger, J. A., Waro, G., Fogelgren, B., Dooley, D. M., Mink, M., and Csiszar, K. (2005) J. Biol. Chem. 280, 22977-22985]. Previous investigations on the expression and purification of recombinant forms of lysyl oxidase [Kagan, H. M., Reddy, V. B., Panchenko, M. V., Nagan, N., Boak, A. M., Gacheru, S. N., and Thomas, K. (1995) J. Cell. Biochem. 59, 329-338] and lysyl oxidase-like proteins [Jung, S. T., Kim, M. S., Seo, J. Y., Kim, H. C., and Kim, Y. (2003) Protein Expression Purif. 31, 240-246] [Molnar, J., Fong, K. S. K., He, Q. P., Hayashi, K., Kim, Y., Fong, S. F. T., Fogelgren, B., Szauter, K. M., Mink, M., and Csiszar, K. (2003) Biochim. Biophys. Acta 1647, 220-224] have been reported in the literature. However, this is the first time that an expression system has been developed yielding sufficient amounts of a recombinant lysyl oxidase for detailed characterization. rDmLOXL-1 is secreted into the medium from S2 cells, and the protein is readily purified by Cibacon blue affinity chromatography yielding 10 mg of protein per liter of medium. The protein, as initially purified, is inactive and has no detectable copper or cofactor present. Following aerobic dialysis against copper, the protein is active and displays an electronic absorption spectrum with lambda(max) at 504 nm, consistent with the presence of an organic cofactor. Addition of phenylhydrazine to the copper-loaded protein produced a high-affinity adduct with lambda(max) at 454 nm. Comparison of the resonance Raman spectra of this adduct and a phenylhydrazine-labeled model compound of lysine tyrosylquinone (LTQ) establishes that the cofactor in the active, copper-containing enzyme is LTQ. Collectively, the data demonstrate that LTQ biogenesis most likely occurs by self-processing chemistry, requiring only the precursor protein, copper, and oxygen. Electron paramagnetic resonance and circular dichroism spectroscopy were used to characterize the Cu(II) site in rDmLOXL-1. The data are consistent with a tetragonal Cu(II) site with nitrogen and oxygen ligands. Recombinant DmLOXL-1 displayed significant activity toward tropoelastin and a wide variety of amines including polyamines and diamines. beta-aminoproprionitrile (betaAPN), a well-known irreversible inhibitor of mammalian lysyl oxidases, is also a potent inhibitor of rDmLOXL-1. Results from this investigation have important implications for the lysyl oxidase family.     

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides: revised kinetic mechanism and kinetics of ATP inhibition

The kinetic mechanisms of the NAD- and NADP-linked reactions catalyzed by glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides were examined using product inhibition, dead-end inhibition and alternate substrate experiments. The results are consistent with a steady-state random mechanism for the NAD-linked and an ordered, sequential mechanism with NADP+ binding first for the NADP-linked reaction. Thus, the enzyme can bind NADP+, NAD+, and glucose 6-phosphate, but the enzyme-glucose 6-phosphate complex can react only with NAD+, not with NADP+. This affects the rate equation for the NADP-linked reaction by introducing a term for a dead-end enzyme-glucose 6-phosphate complex. The kinetic mechanisms represent revisions of those proposed previously (C. Olive, M.E. Geroch, and H.R. Levy, 1971, J. Biol. Chem. 246, 2047-2057) and provide a kinetic basis for the regulation of coenzyme utilization of the enzyme by glucose 6-phosphate concentration (H.R. Levy, and G.H. Daouk, 1979, J. Biol. Chem. 254, 4843-4847) and NADPH/NADP+ concentration ratios (H.R. Levy, G.H. Daouk, and M.A. Katopes, 1979, Arch, Biochem. Biophys. 198, 406-413). The kinetic mechanisms were found to be the same at pH 6.2 and pH 7.8. The kinetics of ATP inhibition of the NAD- and NADP-linked reactions were examined at pH 6.2 and pH 7.8. The results are interpreted in terms of ATP addition to binary enzyme-coenzyme and enzyme-glucose 6-phosphate complexes.     

An investigation on the quinoprotein nature of some fungal and plant oxidoreductases.

The presence of pyrroloquinoline quinone (PQQ) as the organic cofactor of Dactylium dendroides galactose oxidase and lentil (Lens culinaris) seedling amine oxidase, purported PQQ-containing oxidoreductases (Van der Meer, R. A., Jongejan, J. A., and Duine, J. A. (1989) J. Biol. Chem. 264, 7792-7794; Citro, G., Verdina, A., Galati, R., Floris, G., Sabatini, S., and Finazzi-Argo', A. (1989) FEBS Lett. 247, 201-204), was reinvestigated using the nitro blue tetrazolium redoxcycling method (Paz, M. A., Gallop, P. M., Torrelio, B. M., and Flückiger, R. (1988) Biochem. Biophys. Res. Commun. 154, 1330-1337; Paz, M. A., Flückiger, R., Boak, A., Kagan, H. M., and Gallop, P. M. (1991) J. Biol. Chem. 266, 689-692) and the enzyme-linked immunosorbent assay with polyclonal antibodies against PQQ. The possible quinoprotein nature of the laccases from Polyporus versicolor and Rhus vernicifera was also investigated because of the similarities in spectroscopic and kinetic features of these enzymes and the laccase from Phlebia radiata, reported to be a PQQ protein (Karhunen, E., Niku-Paavola, M.-L., Viikari, L., Haltia, T., Van der Meer, R. A., and Duine, J. A. (1990) FEBS Lett. 267, 6-8). The presence of a quinonoid cofactor in lentil seedling amine oxidase is confirmed, whereas galactose oxidase and both laccases do not display any quinoprotein nature.     

Molecular mechanism of calcium channel block by isradipine. Role of a drug-induced inactivated channel conformation

The role of the inactivated channel conformation in the molecular mechanism of Ca(2+) channel block by the 1,4-dihydropyridine (DHP) (+)-isradipine was analyzed in L-type channel constructs (alpha(1Lc); Berjukow, S., Gapp, F., Aczel, S., Sinnegger, M. J., Mitterdorfer, J., Glossmann, H., and Hering, S. (1999) J. Biol. Chem. 274, 6154-6160) and a DHP-sensitive class A Ca(2+) channel mutant (alpha(1A-DHP); Sinnegger, M. J., Wang, Z., Grabner, M., Hering, S., Striessnig, J., Glossmann, H., and Mitterdorfer, J. (1997) J. Biol. Chem. 272, 27686-27693) carrying the high affinity determinants of the DHP receptor site but inactivating at different rates. Ca(2+) channel inactivation was modulated by coexpressing the alpha(1A-DHP)- or alpha(1Lc)-subunits in Xenopus oocytes with either the beta(2a)- or the beta(1a)-subunit and amino acid substitutions in L-type segment IVS6 (I1497A, I1498A, and V1504A). Contrary to a modulated receptor mechanism assuming high affinity DHP binding to the inactivated state we observed no clear correlation between steady state inactivation and Ca(2+) channel block by (+)-isradipine: (i) a 3-fold larger fraction of alpha(1A-DHP)/beta(1a) channels in steady state inactivation at -80 mV (compared with alpha(1A-DHP)/beta(2a)) did not enhance the block by (+)-isradipine; (ii) different steady state inactivation of alpha(1Lc) mutants at -30 mV did not correlate with voltage-dependent channel block; and (iii) the midpoint-voltages of the inactivation curves of slowly inactivating L-type constructs and more rapidly inactivating alpha(1Lc)/beta(1a) channels were shifted to a comparable extent to more hyperpolarized voltages. A kinetic analysis of (+)-isradipine interaction with different L-type channel constructs revealed a drug-induced inactivated state. Entry and recovery from drug-induced inactivation are modulated by intrinsic inactivation determinants, suggesting a synergism between intrinsic inactivation and DHP block.     

A cycling assay for the determination of mono(adenosine-5'-diphosphoribose)-protein bound conjugate levels in mouse tissues

Adenosine-5'-diphosphoribose (ADPR) is quantitatively split into 5'-AMP and ribose phosphate by treatment with alkali at elevated temperature. The 5'-AMP is used to generate NAD through a series of enzyme-catalyzed reactions. The NAD is then determined with a cycling assay modified after E.L. Jacobson and M.K. Jacobson [(1976) Arch. Biochem. Biophys. 175, 627-634]. The specificity of this assay has been verified. With this method the levels of mono(ADPR)-protein bound conjugate in various mouse tissues have been determined.     

The tRNA-interacting factor p43 associates with mammalian arginyl-tRNA synthetase but does not modify its tRNA aminoacylation properties

Arginyl-tRNA synthetase (ArgRS) is one of the nine synthetase components of a multienzyme complex containing three auxiliary proteins as well. We previously established that the N-terminal moiety of the auxiliary protein p43 associates with the N-terminal, eukaryotic-specific polypeptide extension of ArgRS. Because p43 is homologous to Arc1p, a yeast general RNA-binding protein that associates with MetRS and GluRS and plays the role of tRNA-binding cofactor in the aminoacylation reaction, we analyzed the functional significance of p43-ArgRS association. We had previously showed that full-length ArgRS, corresponding to the ArgRS species associated within the multisynthetase complex, and ArgRS with a deletion of 73 N-terminal amino acid residues, corresponding to a free species of ArgRS, both produced in yeast, have similar catalytic parameters (Lazard, M., Kerjan, P., Agou, F., and Mirande, M. (2000) J. Mol. Biol. 302, 991-1004). However, a recent study had suggested that association of p43 to ArgRS reduces the apparent K(M) of ArgRS to tRNA (Park, S. G., Jung, K. H., Lee, J. S., Jo, Y. J., Motegi, H., Kim, S., and Shiba, K. (1999) J. Biol. Chem. 274, 16673-16676). In this study, we analyzed in detail, by gel retardation assays and enzyme kinetics, the putative role of p43 as a tRNA-binding cofactor of ArgRS. The association of p43 with ArgRS neither strengthened tRNA-binding nor changed kinetic parameters in the amino acid activation or in the tRNA aminoacylation reaction. Furthermore, selective removal of the C-terminal RNA-binding domain of p43 from the multisynthetase complex did not affect kinetic parameters for ArgRS. Therefore, p43 has a dual function. It promotes association of ArgRS to the complex via its N-terminal domain, but its C-terminal RNA-binding domain may act as a tRNA-interacting factor for an as yet unidentified component of the complex.     

The motor activity of myosin-X promotes actin fiber convergence at the cell periphery to initiate filopodia formation

Filopodia are actin-rich fingerlike protrusions found at the leading edge of migrating cells and are believed to play a role in directional sensing. Previous studies have shown that myosin-X (myoX) promotes filopodia formation and that this is mediated through its ability to deliver specific cargoes to the cell periphery (Tokuo, H., and M. Ikebe. 2004. Biochem Biophys. Commun. 319:214-220; Zhang, H., J.S. Berg, Z. Li, Y. Wang, P. Lang, A.D. Sousa, A. Bhaskar, R.E. Cheney, and S. Stromblad. 2004. Nat. Cell Biol. 6:523-531; Bohil, A.B., B.W. Robertson, and R.E. Cheney. 2006. Proc. Natl. Acad. Sci. USA. 103:12411-12416; Zhu, X.J., C.Z. Wang, P.G. Dai, Y. Xie, N.N. Song, Y. Liu, Q.S. Du, L. Mei, Y.Q. Ding, and W.C. Xiong. 2007. Nat. Cell Biol. 9:184-192). In this study, we show that the motor function of myoX and not the cargo function is critical for initiating filopodia formation. Using a dimer-inducing technique, we find that myoX lacking its cargo-binding tail moves laterally at the leading edge of lamellipodia and induces filopodia in living cells. We conclude that the motor function of the two-headed form of myoX is critical for actin reorganization at the leading edge, leading to filopodia formation.     

Evaluation by mutagenesis of the importance of 3 arginines in alpha, beta, and gamma subunits of human NAD-dependent isocitrate dehydrogenase

Mammalian NAD-dependent isocitrate dehydrogenase is an allosteric enzyme, activated by ADP and composed of 3 distinct subunits in the ratio 2alpha:1beta:1gamma. Based on the crystal structure of NADP-dependent isocitrate dehydrogenases from Escherichia coli, Bacillus subtilis, and pig heart, and a comparison of their amino acid sequences, alpha-Arg88, beta-Arg99, and gamma-Arg97 of human NAD-dependent isocitrate dehydrogenase were chosen as candidates for mutagenesis to test their roles in catalytic activity and ADP activation. A plasmid harboring cDNA that encodes alpha, beta, and gamma subunits of the human isocitrate dehydrogenase (Kim, Y. O., Koh, H. J., Kim, S. H., Jo, S. H., Huh, J. W., Jeong, K. S., Lee, I. J., Song, B. J., and Huh, T. L. (1999) J. Biol. Chem. 274, 36866-36875) was used to express the enzyme in isocitrate dehydrogenase-deficient E. coli. Wild type (WT) and mutant enzymes (each containing 2 normal subunits plus a mutant subunit with alpha-R88Q, beta-R99Q, or gamma-R97Q) were purified to homogeneity yielding enzymes with 2alpha:1beta:1gamma subunit composition and a native molecular mass of 315 kDa. Specific activities of 22, 14, and 2 micromol of NADH/min/mg were measured, respectively, for WT, beta-R99Q, and gamma-R97Q enzymes. In contrast, mutant enzymes with normal beta and gamma subunits and alpha-R88Q mutant subunit has no detectable activity, demonstrating that, although beta-Arg99 and gamma-Arg97 contribute to activity, alpha-Arg88 is essential for catalysis. For WT enzyme, the Km for isocitrate is 2.2 mm, decreasing to 0.3 mm with added ADP. In contrast, for beta-R99Q and gamma-R97Q enzymes, the Km for isocitrate is the same in the absence or presence of ADP, although all the enzymes bind ADP. These results suggest that beta-Arg99 and gamma-Arg97 are needed for normal ADP activation. In addition, the gamma-R97Q enzyme has a Km for NAD 10 times that of WT enzyme. This study indicates that a normal alpha subunit is required for catalytic activity and alpha-Arg88 likely participates in the isocitrate site, whereas the beta and gamma subunits have roles in the nucleotide functions of this allosteric enzyme.     

Phosphodiesterase-5 Gln817 is critical for cGMP, vardenafil, or sildenafil affinity: its orientation impacts cGMP but not cAMP affinity

The side group of an invariant Gln in cGMP- and cAMP-specific phosphodiesterases (PDE) is held in different orientations by bonds with other amino acids and purportedly discriminates between guanine and adenine in cGMP and cAMP. In cGMP-specific PDE5, Gln(775) constrains the orientation of the invariant Gln(817) side chain, which forms bidentate bonds with 5'-GMP, vardenafil, sildenafil, and 3-isobutyl-1-methylxanthine (IBMX) (Sung, B. J., Hwang, K. Y., Jeon, Y. H., Lee, J. I., Heo, Y. S., Kim, J. H., Moon, J., Yoon, J. M., Hyun, Y. L., Kim, E., Eum, S. J., Park, S. Y., Lee, J. O., Lee, T. G., Ro, S., and Cho, J. M. (2003) Nature 425, 98-102; Huai, Q., Liu, Y., Francis, S. H., Corbin, J. D., and Ke, H. (2004) J. Biol. Chem. 279, 13095-13101; Zhang, K. Y., Card, G. L., Suzuki, Y., Artis, D. R., Fong, D., Gillette, S., Hsieh, D., Neiman, J., West, B. L., Zhang, C., Milburn, M. V., Kim, S. H., Schlessinger, J., and Bollag, G. (2004) Mol. Cell 15, 279-286). PDE5(Q817A) and PDE5(Q775A) were generated to test the hypotheses that Gln(817) is critical for cyclic nucleotide or inhibitor affinity and that Gln(775) immobilizes the Gln(817) side chain to provide cGMP/cAMP selectivity. Allosteric cGMP binding and the molecular mass of the mutant proteins were unchanged compared with PDE5(WT). For PDE5(Q817A), K(m) for cGMP or cAMP was weakened 60- or 2-fold, respectively. For PDE5(Q775A), K(m) for cGMP was weakened approximately 20-fold but was unchanged for cAMP. For PDE5(Q817A), vardenafil, sildenafil, and IBMX inhibitory potencies were weakened 610-, 48-, and 60-fold, respectively, indicating that Gln(817) is a major determinant of potency, especially for vardenafil, and that binding of vardenafil and sildenafil differs substantially. Sildenafil and vardenafil affinity were not significantly affected in PDE5(Q775A). It is concluded that Gln(817) is a positive determinant for PDE5 affinity for cGMP and several inhibitors; Gln(775), which perhaps restricts rotation of Gln(817) side chain, is critical for cGMP affinity but has no measurable effect on affinity for cAMP, sildenafil, or vardenafil.     

Effects of site-directed mutagenesis on structure and function of recombinant rat liver S-adenosylhomocysteine hydrolase. Crystal structure of D244E mutant enzyme

A site-directed mutagenesis, D244E, of S-adenosylhomocysteine hydrolase (AdoHcyase) changes drastically the nature of the protein, especially the NAD(+) binding affinity. The mutant enzyme contained NADH rather than NAD(+) (Gomi, T., Takata, Y., Date, T., Fujioka, M., Aksamit, R. R., Backlund, P. S., and Cantoni, G. L. (1990) J. Biol. Chem. 265, 16102-16107). In contrast to the site-directed mutagenesis study, the crystal structures of human and rat AdoHcyase recently determined have shown that the carboxyl group of Asp-244 points in a direction opposite to the bound NAD molecule and does not participate in any hydrogen bonds with the NAD molecule. To explain the discrepancy between the mutagenesis study and the x-ray studies, we have determined the crystal structure of the recombinant rat-liver D244E mutant enzyme to 2.8-A resolution. The D244E mutation changes the enzyme structure from the open to the closed conformation by means of a approximately 17 degrees rotation of the individual catalytic domains around the molecular hinge sections. The D244E mutation shifts the catalytic reaction from a reversible to an irreversible fashion. The large affinity difference between NAD(+) and NADH is mainly due to the enzyme conformation, but not to the binding-site geometry; an NAD(+) in the open conformation is readily released from the enzyme, whereas an NADH in the closed conformation is trapped and cannot leave the enzyme. A catalytic mechanism of AdoHcyase has been proposed on the basis of the crystal structures of the wild-type and D244E enzymes.     

6-Hydroxycyclohex-1-ene-1-carbonyl-CoA dehydrogenase and 6-oxocyclohex-1-ene-1-carbonyl-CoA hydrolase, enzymes of the benzoyl-CoA pathway of anaerobic aromatic metabolism in the denitrifying bacterium Thauera aromatica.

Benzoyl-CoA is a common intermediate in the anaerobic bacterial metabolism of many aromatic substrates. Two enzymes and ferredoxin of the central benzoyl-CoA pathway in Thauera aromatica have been purified so far. Benzoyl-CoA reductase reduces the aromatic ring with reduced ferredoxin yielding cyclohexa-1,5-diene-1-carbonyl-CoA [Boll, M. & Fuchs, G. (1995) Eur. J. Biochem. 234, 921-933]. Dienoyl-CoA hydratase subsequently adds one molecule of water and thereby produces 6-hydroxycyclohex-1-ene-1-carbonyl-CoA [Laempe, D., Eisenreich, W., Bacher, A., & Fuchs, G. (1998) Eur. J. Biochem. 255, 618-627]. Here two new enzymes, which convert this intermediate to the noncyclic product 3-hydroxypimelyl-CoA, were purified from T. aromatica and studied. 6-Hydroxycyclohex-1-ene-1-carbonyl-CoA dehydrogenase is an NAD(+)-specific beta-hydroxyacyl-CoA dehydrogenase that catalyzes 6-hydroxycyclohex-1-ene-1-carbonyl-CoA + NAD(+) --> 6-oxocyclohex-1-ene-1-carbonyl-CoA + NADH + H(+). 6-Oxocyclohex-1-ene-1-carbonyl-CoA hydrolase acts on the beta-oxoacyl-CoA compound and catalyzes the addition of one molecule of water to the double bound and the hydrolytic C-C cleavage of the alicyclic ring, 6-oxocyclohex-1-ene-1-carbonyl-CoA + 2 H(2)O --> 3-hydroxypimelyl-CoA. The genes for both enzymes, had and oah, were cloned, had was overexpressed in Escherichia coli and the recombinant protein was purified. Hence, presumably all enzymes of the central benzoyl-CoA pathway of anaerobic aromatic metabolism from this organism have now been purified and studied and the corresponding genes have been cloned and sequenced.     

The crystal structure of an HSL-homolog EstE5 complex with PMSF reveals a unique configuration that inhibits the nucleophile Ser144 in catalytic triads

The esterase/lipase family (EC 3.1.1.3/EC 3.1.1.1) represents a diverse group of hydrolases that catalyze the cleavage of ester bonds and are widely distributed in animals, plants and microorganisms. Among these enzymes, hormone-sensitive lipases, play a critical role in the regulation of rodent fat cell lipolysis and are regarded as adipose tissue-specific enzymes. Recently, we reported the structural and biological characterization of EstE5 from the metagenome library [K.H. Nam, M.Y. Kim, S.J. Kim, A. Priyadarshi, W.H. Lee, K.Y. Hwang, Structural and functional analysis of a novel EstE5 belonging to the subfamily of hormone-sensitive lipase, Biochem. Biophys. Res. Commun. 379 (2009) 553-556]. The structure of this protein revealed that it belongs to the HSL-family. Here, we report the inhibition of the activity of the HSL-homolog EstE5 protein as determined by the use of esterase/lipase inhibitors. Our results revealed that the EstE5 protein is significantly inhibited by PMSF. In addition, this is the first study to identify the crystal structures of EstE5-PMSF at 2.4 and 2.5 Å among the HSL-homolog structures. This structural configuration is similar to that adopted when serine proteases are inhibited by PMSF. The results presented here provide valuable information regarding the properties of the HSL-family.     

Identification of a magnesium-dependent NAD(P)(H)-binding domain in the nicotinoprotein methanol dehydrogenase from Bacillus methanolicus

The Bacillus methanolicus methanol dehydrogenase (MDH) is a decameric nicotinoprotein alcohol dehydrogenase (family III) with one Zn(2+) ion, one or two Mg(2+) ions, and a tightly bound cofactor NAD(H) per subunit. The Mg(2+) ions are essential for binding of cofactor NAD(H) in MDH. A B. methanolicus activator protein strongly stimulates the relatively low coenzyme NAD(+)-dependent MDH activity, involving hydrolytic removal of the NMN(H) moiety of cofactor NAD(H) (Kloosterman, H., Vrijbloed, J. W., and Dijkhuizen, L. (2002) J. Biol. Chem. 277, 34785-34792). Members of family III of NAD(P)-dependent alcohol dehydrogenases contain three unique, conserved sequence motifs (domains A, B, and C). Domain C is thought to be involved in metal binding, whereas the functions of domains A and B are still unknown. This paper provides evidence that domain A constitutes (part of) a new magnesium-dependent NAD(P)(H)-binding domain. Site-directed mutants D100N and K103R lacked (most of the) bound cofactor NAD(H) and had lost all coenzyme NAD(+)-dependent MDH activity. Also mutants G95A and S97G were both impaired in cofactor NAD(H) binding but retained coenzyme NAD(+)-dependent MDH activity. Mutant G95A displayed a rather low MDH activity, whereas mutant S97G was insensitive to activator protein but displayed "fully activated" MDH reaction rates. The various roles of these amino acid residues in coenzyme and/or cofactor NAD(H) binding in MDH are discussed.     

Novel pathway for utilization of cyclopropanecarboxylate by Rhodococcus rhodochrous

A new strain isolated from soil utilizes cyclopropanecarboxylate as the sole source of carbon and energy and was identified as Rhodococcus rhodochrous (H. Nishihara, Y. Ochi, H. Nakano, M. Ando, and T. Toraya, J. Ferment. Bioeng. 80:400-402, 1995). A novel pathway for the utilization of cyclopropanecarboxylate, a highly strained compound, by this bacterium was investigated. Cyclopropanecarboxylate-dependent reduction of NAD(+) in cell extracts of cyclopropanecarboxylate-grown cells was observed. When intermediates accumulated in vitro in the absence of NAD(+) were trapped as hydroxamic acids by reaction with hydroxylamine, cyclopropanecarboxohydroxamic acid and 3-hydroxybutyrohydroxamic acid were formed. Cyclopropanecarboxyl-coenzyme A (CoA), 3-hydroxybutyryl-CoA, and crotonyl-CoA were oxidized with NAD(+) in cell extracts, whereas methacrylyl-CoA and 3-hydroxyisobutyryl-CoA were not. When both CoA and ATP were added, organic acids corresponding to the former three CoA thioesters were also oxidized in vitro by NAD(+), while methacrylate, 3-hydroxyisobutyrate, and 2-hydroxybutyrate were not. Therefore, it was concluded that cyclopropanecarboxylate undergoes oxidative degradation through cyclopropanecarboxyl-CoA and 3-hydroxybutyryl-CoA. The enzymes catalyzing formation and ring opening of cyclopropanecarboxyl-CoA were shown to be inducible, while other enzymes involved in the degradation were constitutive.     

From genome to drug lead: identification of a small-molecule inhibitor of the SARS virus

Virtual screening, a fast, computational approach to identify drug leads [Perola, E.; Xu, K.; Kollmeyer, T. M.; Kaufmann, S. H.; Prendergast, F. G. J. Med. Chem.2000, 43, 401; Miller, M. A. Nat. Rev. Drug Disc.2002, 1 220], is limited by a known challenge in crystallographically determining flexible regions of proteins. This approach has not been able to identify active inhibitors of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) using solely the crystal structures of a SARS-CoV cysteine proteinase with a flexible loop in the active site [Yang, H. T.; Yang, M. J.; Ding, Y.; Liu, Y. W.; Lou, Z. Y. Proc. Natl. Acad. Sci. U.S.A.2003, 100, 13190; Jenwitheesuk, E.; Samudrala, R. Bioorg. Med. Chem. Lett.2003, 13, 3989; Rajnarayanan, R. V.; Dakshanamurthy, S.; Pattabiraman, N. Biochem. Biophys. Res. Commun.2004, 321, 370; Du, Q.; Wang, S.; Wei, D.; Sirois, S.; Chou, K. Anal. Biochem.2005, 337, 262; Du, Q.; Wang, S.; Zhu, Y.; Wei, D.; Guo, H. Peptides2004, 25, 1857; Lee, V.; Wittayanarakul, K.; Remsungenen, T.; Parasuk, V.; Sompornpisut, P. Science (Asia)2003, 29, 181; Toney, J.; Navas-Martin, S.; Weiss, S.; Koeller, A. J. Med. Chem.2004, 47, 1079; Zhang, X. W.; Yap, Y. L. Bioorg. Med. Chem.2004, 12, 2517]. This article demonstrates a genome-to-drug-lead approach that uses terascale computing to model flexible regions of proteins, thus permitting the utilization of genetic information to identify drug leads expeditiously. A small-molecule inhibitor of SARS-CoV, exhibiting an effective concentration (EC50) of 23 microM in cell-based assays, was identified through virtual screening against a computer-predicted model of the cysteine proteinase. Screening against two crystal structures of the same proteinase failed to identify the 23-microM inhibitor. This study suggests that terascale computing can complement crystallography, broaden the scope of virtual screening, and accelerate the development of therapeutics to treat emerging infectious diseases such as SARS and Bird Flu.     

The antituberculosis drug ethionamide is activated by a flavoprotein monooxygenase

Ethionamide (ETA), a prodrug that must undergo metabolic activation to exert its cytotoxic effects, is a second line drug against tuberculosis, a disease that infects more than a third of the world's population. It has been proposed, on the basis of genetic experiments, that ETA is activated in Mycobacterium tuberculosis by the protein encoded by the gene Rv3854c (DeBarber, A. E., Mdluli, K., Bosman, M., Bekker, L.-G., and Barry, C. E., III (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 9677-9682; Baulard, A. R., Betts, J. C., Engohang-Ndong, J., Quan, S., McAdam, R. A., Brennan, P. J., Locht, C., and Besra, G. S. (2000) J. Biol. Chem. 275, 28326-28331). We report here the expression, purification, and characterization of the protein encoded by this gene. Our results establish that the enzyme (EtaA) is an FAD-containing enzyme that oxidizes ETA to the corresponding S-oxide. The S-oxide, which has a similar biological activity as ETA, is further oxidized by EtaA to 2-ethyl-4-amidopyridine, presumably via the unstable doubly oxidized sulfinic acid intermediate. This flavoenzyme also oxidizes thiacetazone, thiobenzamide, and isothionicotinamide and thus is probably responsible, as suggested by the observation of crossover resistance, for the oxidative activation of other thioamide antitubercular drugs.     

Cryptobiosis: a new theoretical perspective

The tardigrade is a microscopic creature that under environmental stress conditions undergoes cryptobiosis [Feofilova, E.P., 2003. Deceleration of vital activity as a universal biochemical mechanism ensuring adaptation of microorganisms to stress factors: A review. Appl. Biochem. Microbiol. 39, 1-18; Nelson, D.R., 2002. Current status of the tardigrada: Evolution and ecology. Integrative Comp. Biol. 42, 652-659]-a temporary metabolic depression-which is considered to be a third state between life and death [Clegg, J.S., 2001. Cryptobiosis-a peculiar state of biological organization. Comp. Biochem. Physiol. Part B 128, 613-624]. In contrast with death, cryptobiosis is a reversible state, and as soon as environmental conditions change, the tardigrade "returns to life." Cryptobiosis in general, and among the tardigrade in particular, is a phenomenon poorly understood [Guppy, M., 2004. The biochemistry of metabolic depression: a history of perceptions. Comp. Biochem. Physiol. Part B 139, 435-442; Schill, R.O., et al., 2004. Stress gene (hsp70) sequences and quantitative expression in Milensium tardigradum (Tardigrade) during active and cryptobiotic stages. J. Exp. Biol. 207, 1607-1613; Watanabe, M., et al., 2002. Mechanisn allowing an insect to survive complete dehydration and extreme temperatures. J. Exp. Biol. 205, 2799-2802; Wright, J.C., 2001. Cryptobiosis 300 years on from van Leuwenhoek: what have we learned about tardigrades? Zool. Anz. 240, 563-582]. Moreover, the ability of the tardigrade to bootstrap itself and to return to life seems paradoxical like the legendary Baron von Munchausen who pulled himself out of the swamp by grabbing his own hair. Two theoretical obstacles prevent us from advancing our knowledge of cryptobiosis. First, we lack appropriate theoretical understanding of reversible processes of biological computation in living systems. Second, we lack appropriate theoretical understanding of bootstrapping in living systems. In this short opinion article, I would like to present the idea that although cryptobiosis is obscure from a certain point of view, it makes sense within a scientific perspective suggesting that "organization becomes cause in the matter" [Strohman, R.C., 2000. Organization becomes cause in the matter. Nat. Biotechnol. 18, 575-576]. I present Bateson's idea that organisms have a "recursive hierarchical" form of organization [Neuman, Y., 2004. Meaning making in the immune system. Perspect. Biol. Med. 48, 320-327; Neuman, Y., in press. A theory of meaning. Inform. Sci.] and suggest that this form of organization allows bootstrapping through reversible process of computation as discussed by theoretical physicists [Bennett, C.H., 1982. The thermodynamics of computation-a review. Int. J. Theoret. Phys. 1, 905-940; Landauer and Bennett, 1985].