A Novel Acyl-CoA Beta-Transaminase Characterized from a Metagenome

BACKGROUND: Bacteria are key components in all ecosystems. However, our knowledge of bacterial metabolism is based solely on the study of cultivated organisms which represent just a tiny fraction of microbial diversity. To access new enzymatic reactions and new or alternative pathways, we investigated bacterial metabolism through analyses of uncultivated bacterial consortia. METHODOLOGY/PRINCIPAL FINDINGS: We applied the gene context approach to assembled sequences of the metagenome of the anaerobic digester of a municipal wastewater treatment plant, and identified a new gene which may participate in an alternative pathway of lysine fermentation. CONCLUSIONS: We characterized a novel, unique aminotransferase that acts exclusively on Coenzyme A (CoA) esters, and proposed a variant route for lysine fermentation. Results suggest that most of the lysine fermenting organisms use this new pathway in the digester. Its presence in organisms representative of two distinct bacterial divisions indicate that it may also be present in other organisms

Structural Studies based on two Lysine Dioxygenases with Distinct Regioselectivity Brings Insights Into Enzyme Specificity within the Clavaminate Synthase-Like Family

Abstract Iron(II)/α-ketoacid-dependent oxygenases (αKAOs) are enzymes that catalyze the oxidation of unactivated C-H bonds, mainly through hydroxylation. Among these, those that are active towards amino-acids and their derivatives are grouped in the Clavaminate Synthase Like (CSL) family. CSL enzymes exhibit high regio- and stereoselectivities with strict substrate specificity. This study reports the structural elucidation of two new regiodivergent members, KDO1 and KDO5, active towards lysine, and the structural and computational analysis of the whole family through modelling and classification of active sites. The structures of KDO1 and KDO5 in complex with their ligands show that one exact position in the active site controls the regioselectivity of the reaction. Our results suggest that the substrate specificity and high stereoselectivity typical of this family is linked to a lid that closes up in order to form a sub-pocket around the side chain of the substrate. This dynamic lid is found throughout the family with varying sequence and length and is associated with a conserved stable dimeric interface. Results from this study could be a starting-point for exploring the functional diversity of the CSL family and direct in vitro screening in the search for new enzymatic activities

From structural elucidation to active site classification: exploration of an enzyme family

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Hybrid catalysis: towards an optimal combination of catalysts Application to HMF valorization

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3-keto-5-aminohexanoate Cleavage Enzyme: A Common Fold For An Uncommon Claisen-type Condensation

The exponential increase in genome sequencing output has led to the accumulation of thousands of predicted genes lacking a proper functional annotation. Among this mass of hypothetical proteins, enzymes catalyzing new reactions or using novel ways to catalyze already known reactions might still wait to be identified. Here, we provide a structural and biochemical characterization of the 3-keto-5-aminohexanoate cleavage enzyme (Kce), an enzymatic activity long known as being involved in the anaerobic fermentation of lysine but whose catalytic mechanism has remained elusive so far. Although the enzyme shows the ubiquitous triose phosphate isomerase (TIM) barrel fold and a Zn 2+ cation reminiscent of metal-dependent class II aldolases, our results based on a combination of x-ray snapshots and molecular modeling point to an unprecedented mechanism that proceeds through deprotonation of the 3-keto-5-aminohexanoate substrate, nucleophilic addition onto an incoming acetyl- CoA, intramolecular transfer of the CoA moiety, and final retro-Claisen reaction leading to acetoacetate and 3-aminobutyryl- CoA. This model also accounts for earlier observations showing the origin of carbon atoms in the products, as well as the absence of detection of any covalent acyl-enzyme intermediate. Kce is the first representative of a large family of prokaryotic hypothetical proteins, currently annotated as the "domain of unknown function" DUF849. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.286312739927405Bateman, A., Coggill, P., Finn, R.D., (2010) Acta Crystallogr. F Struct. Biol. Cryst. Commun., 66, pp. 1148-1152Dayhoff, M.O., Schwartz, R., Orcutt, B.C., (1978) Atlas of Protein Sequence and Structure, pp. 345-352. , (Dayhoff, M. O., ed) National Biomedical Research Foundation, Washington, D.CEddy, S.R., (1996) Curr. Opin. Struct. Biol., 6, pp. 361-365Osterman, A., Overbeek, R., (2003) Curr. Opin. Chem. Biol., 7, pp. 238-251Zhang, C., Kim, S.H., (2003) Curr. Opin. Chem. Biol., 7, pp. 28-32Jaroszewski, L., Li, Z., Krishna, S.S., Bakolitsa, C., Wooley, J., Deacon, A.M., Wilson, I.A., Godzik, A., (2009) PLoS Biol., 7, pp. e1000205Kreimeyer, A., Perret, A., Lechaplais, C., Vallenet, D., Médigue, C., Salanoubat, M., Weissenbach, J., (2007) J. Biol. Chem., 282, pp. 7191-7197Barker, H.A., Kahn, J.M., Chew, S., (1980) J. Bacteriol., 143, pp. 1165-1170Barker, H.A., Kahn, J.M., Hedrick, L., (1982) J. Bacteriol., 152, pp. 201-207Yorifuji, T., Jeng, I.M., Barker, H.A., (1977) J. Biol. Chem., 252, pp. 20-31Kabsch, W., (2010) Acta Crystallogr. D Biol. Crystallogr., 66, pp. 125-132(1994) Acta Crystallogr. D Biol. Crystallogr., 50, pp. 760-763. , Collaborative Computational Project, Number 4Sheldrick, G.M., (2008) Acta Crystallogr. A, 64, pp. 112-122Terwilliger, T.C., Berendzen, J., (1999) Acta Crystallogr. D Biol. Crystallogr., 55, pp. 849-861Emsley, P., Lohkamp, B., Scott, W.G., Cowtan, K., (2010) Acta Crystallogr. D Biol. Crystallogr., 66, pp. 486-501Murshudov, G.N., Vagin, A.A., Dodson, E.J., (1997) Acta Crystallogr. D Biol. Crystallogr., 53, pp. 240-255Vagin, A., Teplyakov, A., (1997) J. Appl. Crystallogr., 30, pp. 1022-1025Blanc, E., Roversi, P., Vonrhein, C., Flensburg, C., Lea, S.M., Bricogne, G., (2004) Acta Crystallogr. D Biol. Crystallogr., 60, pp. 2210-2221Chen, V.B., Arendall III, W.B., Headd, J.J., Keedy, D.A., Immormino, R.M., Kapral, G.J., Murray, L.W., Richardson, D.C., (2010) Acta Crystallogr. D Biol. Crystallogr., 66, pp. 12-21Dolinsky, T.J., Nielsen, J.E., McCammon, J.A., Baker, N.A., (2004) Nucleic Acids Res., 32, pp. W665-W667Davis, F.A., Zhang, Y., Andemichael, Y., Fang, T., Fanelli, D.L., Zhang, H., (1999) J. Org. Chem., 64, pp. 1403-1406Trott, O., Olson, A.J., (2010) J. Comput. Chem., 31, pp. 455-461Hinsen, K., (2000) J. Comput. Chem., 21, pp. 79-85Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M., Meng, E.C., Ferrin, T.E., (2004) J. Comput. Chem., 25, pp. 1605-1612Heath, R.J., Rock, C.O., (2002) Nat. Prod. Rep., 19, pp. 581-596Hermann, J.C., Ghanem, E., Li, Y., Raushel, F.M., Irwin, J.J., Shoichet, B.K., (2006) J. Am. Chem. Soc., 128, pp. 15882-15891Hermann, J.C., Marti-Arbona, R., Fedorov, A.A., Fedorov, E., Almo, S.C., Shoichet, B.K., Raushel, F.M., (2007) Nature, 448, pp. 775-779Guillén Schlippe, Y.V., Hedstrom, L., (2005) Arch. Biochem. Biophys., 433, pp. 266-278Burgi, H.B., Dunitz, J.D., Lehn, J.M., Wipff, G., (1974) Tetrahedron, 30, pp. 1563-1572Anstrom, D.M., Kallio, K., Remington, S.J., (2003) Protein Sci., 12, pp. 1822-1832Koon, N., Squire, C.J., Baker, E.N., (2004) Proc. Natl. Acad. Sci. U.S.A., 101, pp. 8295-8300Nagano, N., Orengo, C.A., Thornton, J.M., (2002) J. Mol. Biol., 321, pp. 741-76

Agelasines J, K, and L from the Solomon Islands marine sponge Agelas cf. mauritiana

Three new diterpene alkaloids, agelasine J (3), agelasine K (4), and agelasine L (5), were isolated from the marine sponge Agelas cf. mauritiana collected in the Solomon Islands. The structures of these compounds were elucidated by physical data analyses. They displayed in vitro antimalarial activity against Plasmodium falciparum

Business Incubation in Dar es Salaam

Business incubation is increasingly emphasized on development agendas globally as a tool for entrepreneurship, employment and economic growth. Previous studies focus on comparing European and US business incubators, and outcomes of business incubation in sub-Saharan African settings are comparatively unknown. This paper contributes to the understanding of business incubation in Tanzania by focusing on three themes of business incubation identi fi ed from 43 semi-structured interviews with entrepreneurs and people working with entrepreneurs in Dar es Salaam: 1. The role of the entrepreneur and how it in fl uences business incubation; 2. The business incubator aim and outcome; 3. Perceived constraints for business incubation in Dar es Salaam. Findings include: 1. The variety of entrepreneurship demands additional types of support to generate a valuable contribution; 2. There is a risk that business incubators become excluding organizations, cementing existing roles in society; 3. Business incubators may compensate for constraints on entrepreneurship, but it is important to make them fi t local needs

Revealing the hidden functional diversity of an enzyme family

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