http://www.biochemj.org Biochemical Journal RSS feed -- BJ ChemBio Immediate Publications 0264-6021 1470-8728 Biochemical Journal http://www.biochemj.org/images/BJ_Name.gif http://www.biochemj.org http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20112113 Oleispira antarctica belongs to the PF00756 family of putative esterases, which also includes human esterase D. Here we show that purified recombinant OLEI01171 exhibites high esterase activity against the model esterase substrate α-naphthyl acetate at 5 °C – 30 °C with maximal activity at 15 °C – 20 °C. The esterase activity of OLEI01171 was stimulated 3-8 times by the addition of chloride or several other anions (0.1 M – 1.0 M). Compared to mesophilic PF00756 esterases, OLEI01171 exhibited lower overall protein thermostability. Two crystal structures of OLEI01171 were solved at 1.75 and 2.1 Å resolution and revealed a classical Ser hydrolase catalytic triad and the presence of a chloride or bromide ion bound in the active site close to the catalytic Ser148. Both anions were found to coordinate a potential catalytic water molecule located in the vicinity of the catalytic triad His257. Our data suggest that the bound anion perhaps contributes to the polarization of the catalytic water molecule and increases the rate of the hydrolysis of an acyl-enzyme intermediate. Alanine replacement mutagenesis of OLEI01171 identified ten amino acid residues important for esterase activity. The replacement of Asn225 by Lys had no significant effect on the OLEI01171 activity or thermostability, but resulted in a detectable increase of activity at 35 °C to 45 °C. Our work has provided insight into the molecular mechanisms of activity of a cold-active and anion-activated carboxyl esterase.]]> S Lemak, A Tchigvintsev, P Petit, R Flick, A U. Singer, G Brown, E Evdokimova, O Egorova, C Gonzalez, T N. Chernikova, M M. Yakimov, M Kube, R Reinhardt, P N. Golyshin, A Savchenko, A F. Yakunin 2012-04-23T09:00:00Z doi:10.1042/BJ20112113 Portland Press Limited 2012-04-23 BJ Structure http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120433 K Yamamoto, A Trad, A Baumgart, L Hüske, I Lorenzen, A Chalaris, J Grötzinger, T Dechow, J Scheller, S Rose-John 2012-04-17T15:27:08Z doi:10.1042/BJ20120433 Portland Press Limited 2012-04-17 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120499 Z Zheng, S Itri Amran, J Zhu, O Schmidt-Kittler, K W Kinzler, B Vogelstein, P R Shepherd, P E Thompson, I G Jennings 2012-04-13T13:47:54Z doi:10.1042/BJ20120499 Portland Press Limited 2012-04-13 BJ ChemBio http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111840 O Braten, N Shabek, Y Kravtsova-Ivantsiv, A Ciechanover 2012-04-13T11:23:37Z doi:10.1042/BJ20111840 Portland Press Limited 2012-04-13 BJ ChemBio http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20112103 trans double bond has little influence on the ability of Bax and Bcl-xL to identify and bind to these channels. The stereochemistry of the head group and access to the amide N-H group of ceramide is indispensible for Bax binding indicating that Bax may be interacting with the polar portion of the ceramide channel facing the bulk phase. On the contrary, Bcl-xL binding to ceramide channels is tolerant of stereochemical changes in the head group. This study also revealed that Bcl-xL has an optimal interaction with long-chain ceramides that are elevated early in apoptosis while short-chain ceramides are not well regulated. Inhibitors specific for the hydrophobic groove of Bcl-xL including 2-methoxyantimycin A₃, ABT-737, and ABT-263 provide insights into the region of Bcl-xL involved in binding to ceramide channels. Molecular docking simulations of the lowest energy binding poses of ceramides and Bcl-xL inhibitors to Bcl-xL were consistent with the results of our functional studies and propose potential binding modes.]]> M N Perera, S H Lin, Y K Peterson, A Bielawska, Z M. Szulc, R Bittman, M Colombini 2012-04-11T13:31:33Z doi:10.1042/BJ20112103 Portland Press Limited 2012-04-11 BJ ChemBio http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120408 I Lamberto, H Qin, R Noberini, L Premkumar, C Bourgin, S Riedl, J Song, E B. Pasquale 2012-04-10T14:53:24Z doi:10.1042/BJ20120408 Portland Press Limited 2012-04-10 BJ ChemBio http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20112032 Pseudomonas aeruginosa contains over 20 genes encoding predicted hotdog-fold proteins, none of which have been experimentally characterized. We have found that two P. aeruginosa hotdog proteins display high thioesterase activity against 3-hydroxy-3-methylglutaryl-CoA and glutaryl-CoA (PA5202), and octanoyl-CoA (PA2801). Crystal structures of these proteins were solved (1.70 and 1.75 Å) and revealed a hotdog fold with a potential catalytic carboxylate residue located on the long alpha helix (Asp57 in PA5202 and Glu35 in PA2801). Alanine replacement mutagenesis of PA5202 identified four residues (Asn42, Arg43, Asp57, and Thr76), which are critical for activity and are located in the active site. A P. aeruginosa PA5202 deletion strain showed an increased secretion of the antimicrobial pigment pyocyanine and an increased expression of genes involved in pyocyanin biosynthesis suggesting a functional link between the PA5202 activity and pyocyanin production. Thus, the P. aeruginosa hotdog thioesterases PA5202 and PA2801 have similar structures, but exhibit different substrate preferences and functions.]]> C Gonzalez, A Tchigvintsev, G Brown, R Flick, E Evdokimova, X Xu, J Osipiuk, M Cuff, S Lynch, A Joachimiak, A Savchenko, A F. Yakunin 2012-03-23T10:59:46Z doi:10.1042/BJ20112032 Portland Press Limited 2012-03-23 BJ Structure http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120036 Bryum coronatum, is an endo-acting enzyme that hydrolyzes glycosidic bonds of chitin, a β-1,4-linked polysacharide of N-acetylglucosamine, (GlcNAc)_n, through an inverting mechanism. When the wild-type enzyme was incubated with alpha-(GlcNAc)₂ fluoride [alpha-(GlcNAc)₂-F] in the absence or presence of (GlcNAc)₂, (GlcNAc)₂ and hydrogen fluoride were found to be produced through the Hehre resynthesis-hydrolysis mechanism. To convert BcChi-A into a glycosynthase, we employed the strategy reported by Honda et al. (J. Biol. Chem. 281, 1426-1431 (2006); Glycobiology, 18, 325-330 (2008)); that is, Ser102 holding a nucleophilic water molecule and Glu70 acting as a catalytic base were mutated, producing S102A, S102C, S102D, S102G, S102H, S102T, E70G, and E70Q. In all mutated enzymes except S102T, hydrolytic activity toward (GlcNAc)₆ was not detected under the conditions we employed. Among the inactive BcChi-A mutants, S102A, S102C, S102G, and E70G were found to successfully synthesize (GlcNAc)₄ as a major product from alpha-(GlcNAc)₂-F in the presence of (GlcNAc)₂. The S102A mutant showed the greatest glycosynthase activity due to its enhanced F^- releasing activity and its suppressed hydrolytic activity. This is the first report on a glycosynthase that employs amino sugar fluoride as a donor substrate.]]> T Ohnuma, T Fukuda, S Dozen, Y Honda, M Kitaoka, T Fukamizo 2012-03-21T14:14:03Z doi:10.1042/BJ20120036 Portland Press Limited 2012-03-21 BJ Structure http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20112219 I C Martins, F Gomes-Neto, A F Faustino, F A Carvalho, F A Carneiro, P T Bozza, R Mohana-Borges, M A Castanho, F C Almeida, N C. Santos, A T Da Poian 2012-03-19T14:50:05Z doi:10.1042/BJ20112219 Portland Press Limited 2012-03-19 BJ ChemBio