anti-virulent-disruption-pathogenic-biofilms-using-engineered-quorum

Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases

The rapid emergence of multi-drug resistant bacteria has accelerated the need for novel therapeutic approaches to counter life-threatening infections. The ...

University of Utah

Cytokinesis in all organisms involves the creation of membranous barriers that demarcate individual daughter cells. In fission yeast, a signaling module termed the septation initiation network (SIN) plays an essential role in the assembly of new membranes and cell wall during cytokinesis. In this study, we have characterized Slk1p, a protein-kinase related to the SIN component Sid2p. Slk1p is expressed specifically during meiosis and localizes to the spindle pole bodies (SPBs) during meiosis I and II in a SIN-dependent manner. Slk1p also localizes to the forespore membrane during sporulation. Cells lacking Slk1p display defects associated with sporulation, leading frequently to the formation of asci with smaller and/or fewer spores. The ability of slk1 Delta cells to sporulate, albeit inefficiently, is fully abolished upon compromise of function of Sid2p, suggesting that Slk1p and Sid2p play overlapping roles in sporulation. Interestingly, increased expression of the syntaxin Psy1p rescues the sporulation defect of sid2-250 slk1 Delta. Thus, it is likely that Slk1p and Sid2p play a role in forespore membrane assembly by facilitating recruitment of components of the secretory apparatus, such as Psy1p, to allow membrane expansion. These studies thereby provide a novel link between the SIN and vesicle trafficking during cytokinesis.

The meiosis-specific Sid2p-related protein Slk1p regulates forespore membrane assembly in fission yeast.

The PLL(PTE-like lactonase)-group of enzymes within the amidohydrolase superfamily hydrolyze N-acyl-homoserine lactones (AHLs) that are involved in bacterial quorum-sensing pathways. These enzymes possess the (beta/alpha)(8)-barrel fold and serve as attractive templates for in vitro evolution and engineering of quorum-quenching biological molecules that can serve as antivirulence therapeutic agents. Using a quorum-quenching lactonase from Mycobacterium avium subsp. paratuberculosis K-10 (GI: 41409766) as the initial template for in vitro evolution experiments, we enhanced the catalytic efficiency and increased the substrate range of the wild-type enzyme through a single point mutation on the loop at the C-terminal end of the eighth beta-strand. This N266Y mutant had an increased value of k(cat)/K(M) of 30- and 32-fold toward 3-oxo-N-octanoyl-l-homoserine lactone and N-hexanoyl-l-homoserine lactone, respectively; the evolved mutant also exhibited lactonase activity toward 3-oxo-N-hexanoyl-l-homoserine lactone and N-butyryl-l-homoserine lactone, AHLs that were previously not hydrolyzed by the wild-type enzyme. This article reinforces the evolutionary potential of the (beta/alpha)(8)-barrel fold and highlights the possibility of using quorum-quenching lactonases in the amidohydrolase superfamily as templates for engineering biomolecules of therapeutic use.

Directed evolution of a quorum-quenching lactonase from Mycobacterium avium subsp. paratuberculosis K-10 in the amidohydrolase superfamily.

A thermostable quorum-quenching lactonase from Geobacillus kaustophilus HTA426 (GI: 56420041) was used as an initial template for in vitro directed evolution experiments. This enzyme belongs to the phosphotriesterase-like lactonase (PLL) group of enzymes within the amidohydrolase superfamily that hydrolyze N-acylhomoserine lactones (AHLs) that are involved in virulence pathways of quorum-sensing pathogenic bacteria. Here we have determined the N-butyryl-L-homoserine lactone-liganded structure of the catalytically inactive D266N mutant of this enzyme to a resolution of 1.6 Å. Using a tunable, bioluminescence-based quorum-quenching molecular circuit, the catalytic efficiency was enhanced, and the AHL substrate range increased through two point mutations on the loops at the C-terminal ends of the third and seventh β-strands. This E101N/R230I mutant had an increased value of k(cat)/K(m) of 72-fold toward 3-oxo-N-dodecanoyl-L-homoserine lactone. The evolved mutant also exhibited lactonase activity toward N-butyryl-L-homoserine lactone, an AHL that was previously not hydrolyzed by the wild-type enzyme. Both the purified wild-type and mutant enzymes contain a mixture of zinc and iron and are colored purple and brown, respectively, at high concentrations. The origin of this coloration is suggested to be because of a charge transfer complex involving the β-cation and Tyr-99 within the enzyme active site. Modulation of the charge transfer complex alters the lactonase activity of the mutant enzymes and is reflected in enzyme coloration changes. We attribute the observed enhancement in catalytic reactivity of the evolved enzyme to favorable modulations of the active site architecture toward productive geometries required for chemical catalysis.

Directed evolution of a thermostable quorum-quenching lactonase from the amidohydrolase superfamily.

Polyketides are chemically diverse and medicinally important biochemicals that are biosynthesized from acyl-CoA precursors by polyketide synthases. One of the limitations to combinatorial biosynthesis of polyketides has been the lack of a toolkit that describes the means of delivering novel acyl-CoA precursors necessary for polyketide biosynthesis. Using five acid-CoA ligases obtained from various plants and microorganisms, we biosynthesized an initial library of 79 acyl-CoA thioesters by screening each of the acid-CoA ligases against a library of 123 carboxylic acids. The library of acyl-CoA thioesters includes derivatives of cinnamyl-CoA, 3-phenylpropanoyl-CoA, benzoyl-CoA, phenylacetyl-CoA, malonyl-CoA, saturated and unsaturated aliphatic CoA thioesters, and bicyclic aromatic CoA thioesters. In our search for the biosynthetic routes of novel acyl-CoA precursors, we discovered two previously unreported malonyl-CoA derivatives (3-thiophenemalonyl-CoA and phenylmalonyl-CoA) that cannot be produced by canonical malonyl-CoA synthetases. This report highlights the utility and importance of determining substrate promiscuities beyond conventional substrate pools and describes novel enzymatic routes for the establishment of precursor-directed combinatorial polyketide biosynthesis.

Establishing a toolkit for precursor-directed polyketide biosynthesis: exploring substrate promiscuities of acid-CoA ligases.

&#x4F53;&#x5916;&#x8FDB;&#x5316;&#x548C;&#x589E;&#x5F3A;&#x6D3B;&#x6027;&#x4E0E;&#x4EF2;&#x88C1;&#x6DEC;&#x706B; lactonases &#x5DE5;&#x7A0B;&#x53D6;&#x5F97;&#x4E86;&#x4F5C;&#x4E3A;&#x6A21;&#x677F;&#xFF0C;&#x4EA7;&#x751F; E101G/R230C GKL &#x7A81;&#x53D8;&#x4E0E;&#x50AC;&#x5316;&#x6D3B;&#x52A8;&#x589E;&#x52A0;&#x548C;&#x6269;&#x5927;&#x7684;&#x57FA;&#x677F;&#x8303;&#x56F4;&#x4F7F;&#x7528;&#x4E00;&#x79CD;&#x8010;&#x9AD8;&#x6E29;&#x7684; GKL &#x9176; [&#x5468;&#x6881;&#x6DD1;&#x6021;&#x8BAE;&#x5458;&#x3001; J.Y.&#x3001; &#x859B;&#xFF0C;B.&#xFF0C;&#x674E;&#x3001; K.H.&#x3001; &#x8463;&#xFF0C;A.&#x3001; &#x90AC; L.&#x3001; &#x7F57;&#x5BBE;&#x900A;&#x3001; R.C.&#x7EA2;&#x8C46;&#x6749;&#xFF0C;W.S.(2010 &#x5E74;) J &#x5DE5;&#x4E1A;&#x56ED;&#x6D77;&#x535A; Chem 285&#xFF0C;40911 40920]&#x3002;&#x8FD9;&#x79CD;&#x9176;&#x62E5;&#x6709; (&beta;/&alpha;) 8 &#x6876;&#x6298;&#x53E0;&#xFF0C;&#x5E76;&#x4E14;&#x662F;&#x9501;&#x76F8;&#x73AF; (Phosphotriesterase-Like &#x4E1D;&#x6C28;&#x9178;&#x5185;&#x916F;&#x9176;&#xFF09; &#x7684;&#x6210;&#x5458;-&#x7EC4;&#x7684;&#x9176;&#x6C34;&#x89E3;&#x9176;&#x5BB6;&#x65CF;&#x5185;&#x6C34;&#x89E3; N-&#x9170;&#x57FA;&#x9AD8;&#x4E1D;&#x6C28;&#x9178;&#x5185;&#x916F;&#xFF0C;&#x5176;&#x4E2D;&#x8C03;&#x89E3;&#x7684;&#x7EC6;&#x83CC;&#x7FA4;&#x4F53;&#x611F;&#x5E94;&#x7684;&#x9014;&#x5F84;&#x3002;&#x7ED3;&#x6784;&#x7684;&#x8FDB;&#x5316; N-&#x4E01;&#x9170;-L-&#x9AD8;&#x4E1D;&#x6C28;&#x9178;&#x5185;&#x916F; (&#x886C;&#x5E95;)-&#x7ED1;&#x5B9A;&#x7684; E101G/R230C GKL &#x9176;&#x5F97;&#x5230;&#x89E3;&#x51B3;&#xFF0C;&#x5728;&#x573A;&#x7684;&#x706D;&#x6D3B;&#x7684; D266N &#x7A81;&#x53D8;&#xFF0C;&#x51B3;&#x8BAE;&#x5C06; 2.2 &#x6210;&#x63D0;&#x4F9B;&#x89E3;&#x91CA;&#x89C2;&#x5BDF;&#x5230;&#x7684;&#x7387;&#x589E;&#x5F3A;&#x529F;&#x80FD; &#xFF1B;&#x6B64;&#x5916;&#xFF0C;&#x9530;&#x91CD;&#x7EC4;&#x9176;&#x7684;&#x50AC;&#x5316;&#x975E;&#x6D3B;&#x52A8;&#x7684; E101N/D266N &#x7A81;&#x53D8;&#x7684;&#x57FA;&#x677F;&#x7ED1;&#x5B9A;&#x7ED3;&#x6784;&#x5DF2;&#x83B7;&#x5F97;&#x5BF9;&#x4E00;&#x9879;&#x51B3;&#x8BAE;&#x7684; 2.1 &#x6210; &#xFF1B;&#x65E0;&#x914D;&#x4F53;&#x3001; &#x9530;&#x91CD;&#x7EC4; E101N &#x7A81;&#x53D8;&#x7684; 2.6 &#x7684;&#x51B3;&#x8BAE;&#x5C06;&#x6210; &#xFF1B;&#x65E0;&#x914D;&#x4F53;&#x7684;&#x950C;&#x91CD;&#x7EC4;&#x91CE;&#x751F;&#x578B;&#x7ED3;&#x6784;&#x3001; E101N&#x3001; R230D&#x3001; E101G/R230C &#x7A81;&#x53D8;&#x4F53; GKL 2.1 &#x7684;&#x51B3;&#x8BAE;&#x548C;&#x57C3;&#x3001; 2.1 &#x6210;&#xFF0C;1.9 &#x6210;&#xFF0C;&#x548C; 2.0 &#x6210;&#xFF0C;&#x5206;&#x522B;&#x3002;&#x5C24;&#x5176;&#x662F;&#xFF0C;E101G/R230C &#x8FDB;&#x5316;&#x7A81;&#x53D8;&#x4F53;&#x7684; GKL &#x7ED3;&#x6784;&#x63D0;&#x4F9B;&#x4E86;&#x8BC1;&#x636E;&#x6709;&#x52A9;&#x4E8E;&#x89C2;&#x5BDF;&#x5230;&#x52A0;&#x5F3A;&#x5728;&#x50AC;&#x5316;&#x53CD;&#x5E94;&#x4E2D;&#x7684;&#x50AC;&#x5316;&#x751F;&#x4EA7;&#x6D3B;&#x52A8;&#x7F51;&#x7AD9;&#x4F53;&#x7CFB;&#x7ED3;&#x6784;&#x3002;&#x6D53;&#x5EA6;&#x9AD8;&#xFF0C;&#x91CE;&#x751F;&#x578B;&#x53CA;&#x7A81;&#x53D8;&#x578B; GKL &#x9176;&#x6709;&#x5DEE;&#x5F02;&#xFF0C;&#x4E0E;&#x7740;&#x8272;&#x5438;&#x5149;&#x5EA6;&#x6700;&#x5927;&#x503C;&#x8303;&#x56F4;&#x4E2D;&#x7684; 512 &#x5230; 553 nm nm&#x3002;&#x91CE;&#x751F;&#x578B;&#x53CA;&#x7A81;&#x53D8;&#x578B; GKL &#x7684;&#x7ED3;&#x6784;&#x63D0;&#x4F9B;&#x4E4B;&#x95F4;&#xFF0C;&#x7740;&#x8272;&#x7684;&#x8D77;&#x6E90;&#x548C;&#x9176;&#x7684;&#x6D3B;&#x6027;&#x90E8;&#x4F4D;&#x5185; &alpha;-&#x9633;&#x79BB;&#x5B50;&#x548C; Tyr99 &#x4E4B;&#x95F4;&#x7684;&#x7535;&#x8377;&#x8F6C;&#x79FB;&#x7EDC;&#x5408;&#x7269;&#x6E29;&#x987A;&#x7684;&#x94FE;&#x63A5;&#x3002;&#x4E24;&#x8005;&#x5408;&#x8BA1;&#xFF0C;&#x8FD9;&#x9879;&#x7814;&#x7A76;&#x63D0;&#x4F9B;&#x4E86;&#x8BC1;&#x636E;&#x7684;&#x9176;&#x50AC;&#x5316;&#x9176;&#x6D3B;&#x52A8;&#x7F51;&#x7AD9;&#x4F53;&#x7CFB;&#x7ED3;&#x6784;&#x4E2D;&#x7684;&#x5FAE;&#x5999;&#x53D8;&#x5316;&#x901A;&#x8FC7; modulability&#x3002;

&#x4E1D;&#x6C28;&#x9178;&#x5185;&#x8FDB;&#x5316;&#x4EF2;&#x88C1;&#x6DEC;&#x706B; GKL &#x916F;&#x9176;&#x7ED3;&#x6784;&#x8BC1;&#x636E;&#x7684;&#x4E00;&#x79CD;&#x751F;&#x4EA7;&#x6027;&#x6D3B;&#x52A8;&#x7F51;&#x7AD9;&#x4F53;&#x7CFB;&#x7ED3;&#x6784;&#x3002;

The in vitro evolution and engineering of quorum-quenching lactonases with enhanced reactivities was achieved using a thermostable GKL enzyme as template, yielding the E101G/R230C GKL mutant with increased catalytic activity and broadened substrate range [Chow, J. Y., Xue, B., Lee, K. H., Tung, A., Wu, L., Robinson, R. C., and Yew, W. S. (2010) J Biol Chem 285, 40911-40920]. This enzyme possesses the (Î²/Î±)8-barrel fold and is a member of the PLL (Phosphotriesterase-Like Lactonase)-group of enzymes within the amidohydrolase superfamily that hydrolyze N-acyl-homoserine lactones, which mediate the quorum-sensing pathways of bacteria. The structure of the evolved N-butyryl-L-homoserine lactone (substrate)-bound E101G/R230C GKL enzyme was solved, in the presence of the inactivating D266N mutation, to a resolution of 2.2 Ã… to provide an explanation for the observed rate enhancements; in addition, the substrate-bound structure of the catalytically-inactive E101N/D266N mutant of the manganese-reconstituted enzyme was obtained to a resolution of 2.1 Ã…; the ligand-free, manganese-reconstituted E101N mutant to a resolution of 2.6 Ã…; and the structures of ligand-free zinc-reconstituted wild-type, E101N, R230D, and E101G/R230C mutants of GKL to a resolution of 2.1 Ã…, 2.1 Ã…, 1.9 Ã…, and 2.0 Ã…, respectively. In particular, the structure of the evolved E101G/R230C mutant of GKL provides evidence for a catalytically productive active site architecture that contributes to the observed enhancement in catalysis. At high concentrations, wild-type and mutant GKL enzymes are differentially colored, with absorbance maximums in the range of 512 nm to 553 nm. The structures of the wild-type and mutant GKL provide a tractable link between the origins of the coloration and the charge-transfer complex between the Î±-cation and Tyr99 within the enzyme active site. Taken together, this study provides evidence for the modulability of enzymatic catalysis through subtle changes in enzyme active site architecture.

Structural Evidence of a Productive Active Site Architecture for an Evolved Quorum-quenching GKL Lactonase.

L&#39;&eacute;volution in vitro et l&#39;ing&eacute;nierie de quorum-trempe lactonases avec r&eacute;activit&eacute; am&eacute;lior&eacute;e a &eacute;t&eacute; obtenue en utilisant une enzyme GKL thermostable comme mod&egrave;le, ce qui donne le mutant GKL E101G/R230C avec augmentation de l&#39;activit&eacute; catalytique et la gamme &eacute;largie de substrat [Chow, J. Y., Xue, B., Lee, K. H., Tung, A., Wu, L., Robinson, R. C. et l&#39;if, W. S. (2010) J Biol Chem 285, 40911-40920]. Cette enzyme poss&egrave;de le pli de 8-baril (&beta;/&alpha;) et est membre de la PLL (Phosphotriesterase-Like Lactonase)-groupe d&#39;enzymes dans la superfamille amidohydrolase qui hydrolysent les N-acyl-homos&eacute;rine lactones, qui n&eacute;gocient les voies quorum-sensing de bact&eacute;ries. La structure de l&#39;&eacute;volu&eacute;e N-butyryl-L-homos&eacute;rine lactone (substrat)-enzyme de E101G/R230C GKL li&eacute; a &eacute;t&eacute; r&eacute;solu, en pr&eacute;sence de la mutation inactivant de D266N, &agrave; une r&eacute;solution de 2,2 &Aring; de fournir une explication pour les am&eacute;liorations de la vitesse observ&eacute;e ; en outre, la structure du substrat-bondissent du mutant catalytiquement inactif E101N/D266N de l&#39;enzyme de mangan&egrave;se-reconstitu&eacute;e a &eacute;t&eacute; obtenue &agrave; une r&eacute;solution de 2,1 &Aring; ; le mutant E101N ligand libre, mangan&egrave;se-reconstitu&eacute;e &agrave; une r&eacute;solution de 2.6 &Aring; ; et les structures des ligands libres zinc-reconstitu&eacute;e sauvage, mutants E101N, R230D et E101G/R230C de GKL pour une r&eacute;solution de 2,1 &Aring;, 2,1 &Aring;, 1,9 &Aring; et 2.0 &Aring;, respectivement. En particulier, la structure du mutant E101G/R230C &eacute;volu&eacute;e de GKL fournit des preuves pour une architecture catalytiquement productives site actif qui contribue &agrave; l&#39;augmentation observ&eacute;e dans la catalyse. &Agrave; forte concentration, sauvage et les mutants des enzymes GKL sont color&eacute;s diff&eacute;remment, avec maxima d&#39;absorbance dans la gamme de 512 nm &agrave; 553 nm. Les structures de la sauvage et mutante GKL assurent une liaison souple entre les origines de la coloration et le complexe de transfert de charge entre le &alpha;-cation et Tyr99 dans le site actif de l&#39;enzyme. Pris ensemble, cette &eacute;tude d&eacute;montre pour la modulability de la catalyse enzymatique gr&acirc;ce &agrave; des changements subtils dans l&#39;architecture du site actif enzymatique.

Preuves de structure d&#39;une Architecture de Site actif productif pour un Lactonase GKL Quorum-trempe &eacute;volu&eacute;e.

Die in-vitro-Evolution und Engineering der Quorum-abschrecken Lactonases mit verst&auml;rkten Reactivities wurde erreicht mit einem hitzebest&auml;ndigen GKL-Enzym als Vorlage, woraus sich der E101G/R230C GKL-Mutant mit erh&ouml;hten katalytische Aktivit&auml;t und erweiterten Substrat [Chow, J. Y., Xue, B., Lee, K. H., Tung, A., Wu, L., Robinson, R. C. und Eibe, W. S. (2010) J Biol Chem 285, 40911-40920]. Dieses Enzym besitzt die (&beta;/&alpha;)-8-Fass-Klappe und ist Mitglied der PLL (Phosphotriesterase-Like-Lactonase)-Gruppe von Enzymen innerhalb der &Uuml;berfamilie Amidohydrolase, die N-Acyl-Homoserin Lactone, hydrolysieren die die Quorum-sensing-Bahnen von Bakterien zu vermitteln. Die Struktur der weiterentwickelte N-Butyryl-L-Homoserin-lacton (Substrat)-gebundenen E101G/R230C GKL Enzym wurde gel&ouml;st, in Anwesenheit der inaktivierende D266N-Mutation, einer Aufl&ouml;sung von 2.2 &Aring;, eine Erkl&auml;rung f&uuml;r die beobachteten Preise-Erweiterungen; Zus&auml;tzlich wurde Substrat gebundene Struktur der katalytisch inaktive E101N/D266N-Mutant des Enzyms Mangan-rekonstituiert gewonnen, einer Aufl&ouml;sung von 2,1 &Aring;; der Ligand-frei, Mangan-rekonstituiert E101N Mutant mit einer Aufl&ouml;sung von 2.6 &Aring;; und die Strukturen der Ligand-freie Zink-rekonstituiert Wildtyp, E101N, R230D und E101G/R230C Mutanten der GKL auf eine Aufl&ouml;sung von 2,1 &Aring;, 2.1 &Aring;, 1,9 &Aring;, und 2,0 &Aring;, beziehungsweise. Insbesondere stellt die Struktur des den weiterentwickelten E101G/R230C-Mutanten der GKL Beweise f&uuml;r eine katalytisch produktive aktiven Site-Architektur, die zu den beobachteten Erweiterung in Katalyse beitr&auml;gt. In hohen Konzentrationen, Wildtyp und Mutanten GKL Enzyme sind differentiell gef&auml;rbt, mit Absorption Maximum im Bereich von 512 nm bis 553 nm. Die Strukturen der Wildtyp und Mutanten GKL enthalten steuerbar Links zwischen den Urspr&uuml;ngen der F&auml;rbung und der Charge-Transfer-Komplex zwischen dem &alpha;-kation und Tyr99 innerhalb der Enzym-aktiv-Website. Zusammen genommen bietet diese Studie Beweise f&uuml;r die Modulierbarkeit enzymatische Katalyse durch subtile Ver&auml;nderungen in Enzym aktiven Website-Architektur.

Strukturelle Beweise f&uuml;r eine produktive aktiven Site-Architektur f&uuml;r ein weiterentwickelter Quorum-abschrecken GKL-Lactonase.

L&#39;evoluzione in vitro e ingegneria del quorum-tempra lactonases con una maggiore reattivit&agrave; &egrave; stato realizzato utilizzando un enzima termostabile GKL come modello, producendo il mutante GKL E101G/R230C con aumento dell&#39;attivit&agrave; catalitica e substrato ampliata gamma [Chow, J. Y., Xue, B., Lee, K. H., Tung, A., Wu, L., Robinson, R. C. e Yew, S. (2010) J Biol Chem 285, 40911-40920]. Questo enzima possiede la piega 8-canna (&alpha;/&beta;) ed &egrave; membro del PLL (Phosphotriesterase-Like Lactonase)-gruppo di enzimi all&#39;interno della superfamiglia amidohydrolase in grado di idrolizzare N-acil-omoserina lattoni, che mediano i quorum sensing percorsi di batteri. La struttura dell&#39;evoluta N-butirril-L-omoserina lattone (substrato)-enzima E101G/R230C GKL associato &egrave; stato risolto, in presenza della mutazione D266N inattivanti, a una risoluzione di 2.2 &Aring; per fornire una spiegazione per i miglioramenti osservati tasso; Inoltre, la struttura associato a substrato del mutante E101N/D266N cataliticamente inattivo dell&#39;enzima manganese-ricostituito &egrave; stata ottenuta per una risoluzione di 2.1 &Aring;; il ligando-free, manganese-ricostituito E101N mutante a una risoluzione di 2,6 &Aring;; e le strutture di ligando libero zinco-ricostituito wild-type, E101N, R230D ed E101G/R230C mutanti di GKL per una risoluzione di 2.1 &Aring;, 2.1 &Aring;, 1.9 &Aring; e 2.0 &Aring;, rispettivamente. In particolare, la struttura del mutante E101G/R230C evoluta di GKL fornisce la prova per un&#39;architettura del sito attivo cataliticamente produttivo che contribuisce al miglioramento osservato nella catalisi. Ad alte concentrazioni, wild-type e mutanti enzimi GKL differenzialmente sono colorati, con valori massimi di assorbimento nell&#39;intervallo di 512 nm a 553 nm. Le strutture della wild-type e mutanti GKL forniscono un collegamento gestibile tra le origini della colorazione e il complesso di trasferimento di carica tra il &alpha;-cazione e Tyr99 all&#39;interno del sito attivo enzimatico. Presi insieme, questo studio fornisce la prova per la modulabilit&agrave; della catalisi enzimatica attraverso sottili cambiamenti nell&#39;architettura del sito attivo enzimatico.

Prove strutturali di un&#39;architettura produttiva sito attivo per un evoluto Quorum-tempra GKL Lactonase.

&#x30AF;&#x30A9;&#x30FC;&#x30E9;&#x30E0;&#x713C; lactonases &#x3067;&#x5F37;&#x5316;&#x3055;&#x308C;&#x305F;&#x53CD;&#x5FDC;&#x6027;&#x306E; in vitro &#x9032;&#x5316;&#x5DE5;&#x5B66; GKL &#x9175;&#x7D20;&#x89E6;&#x5A92;&#x6D3B;&#x52D5;&#x306E;&#x5897;&#x52A0;&#x3068;&#x62E1;&#x5927;&#x57FA;&#x677F;&#x7BC4;&#x56F2; E101G/R230C GKL &#x5909;&#x7570;&#x3092;&#x964D;&#x4F0F;&#x30C6;&#x30F3;&#x30D7;&#x30EC;&#x30FC;&#x30C8;&#x3068;&#x3057;&#x3066;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x9054;&#x6210;&#x3055;&#x308C;&#x305F; [&#x30C1;&#x30E7;&#x30A6;&#x3001;j., &#x96EA;&#x3001;B &#x306E;&#x6850;&#x3001;&#x5065;&#xFF0C; &#x674E; A.&#x3001;&#x5449;&#x3001;l.&#x3001;&#x30ED;&#x30D3;&#x30F3;&#x30BD;&#x30F3;&#x3001;r. c.&#x3001;&#x30A4;&#x30C1;&#x30A4;&#x3001;W. S. &#xFF08;2010&#xFF09; J &#x751F;&#x7269;&#x5316;&#x5B66; 285&#x3001;40911 40920]&#x3002;&#x3053;&#x306E;&#x9175;&#x7D20; (&beta;/&alpha;) 8 &#x30D0;&#x30EC;&#x30EB;&#x500D;&#x3092;&#x6240;&#x6709;&#x3057;&#x3066;&#x3044;&#x308B;&#x3057;&#x3001;PLL (Phosphotriesterase-Like Lactonase) &#x306E;&#x30E1;&#x30F3;&#x30D0;&#x30FC;&#x3067;&#x3042;&#x308B;-&#x7D30;&#x83CC;&#x306E;&#x30AF;&#x30AA;&#x30E9;&#x30E0;&#x30BB;&#x30F3;&#x30B7;&#x30F3;&#x30B0;&#x7D4C;&#x8DEF;&#x3092;&#x4EF2;&#x4ECB;&#x3059;&#x308B; N &#x30A2;&#x30B7;&#x30EB; &#x30DB;&#x30E2;&#x30BB;&#x30EA;&#x30F3;&#x306E;&#x88FD;&#x9020;&#x30E9;&#x30AF;&#x30C8;&#x30F3;&#x52A0;&#x6C34;&#x5206;&#x89E3;&#x9175;&#x7D20;&#x30A2;&#x30DF;&#x30C9;&#x30D2;&#x30C9;&#x30ED;&#x30E9;&#x30FC;&#x30BC; &#x30B9;&#x30FC;&#x30D1;&#x30FC;&#x30D5;&#x30A1;&#x30DF;&#x30EA;&#x30FC;&#x5185;&#x306E;&#x30B0;&#x30EB;&#x30FC;&#x30D7;&#x3002;&#x9032;&#x5316;&#x3057;&#x305F; N-&#x30D6;&#x30C1;&#x30EA;&#x30EB;-L d-&#x30DB;&#x30E2;&#x30BB;&#x30EA;&#x30F3;&#x306E;&#x88FD;&#x9020;&#x30E9;&#x30AF;&#x30C8;&#x30F3; &#xFF08;&#x57FA;&#x677F;&#xFF09; &#x306E;&#x69CB;&#x9020;-&#x30D0;&#x30A4;&#x30F3;&#x30C9;&#x3055;&#x308C;&#x305F; E101G/R230C GKL &#x9175;&#x7D20;&#x89E3;&#x6C7A; 2.2 &#x306E;&#x89E3;&#x50CF;&#x5EA6;&#x306E;&#x4E0D;&#x6D3B;&#x5316; D266N &#x306E;&#x7A81;&#x7136;&#x5909;&#x7570;&#x306E;&#x5B58;&#x5728;&#x4E0B;&#x3067; &Aring; &#x89B3;&#x6E2C;&#x91CF;&#x306E;&#x5F37;&#x5316;; &#x306E;&#x8AAC;&#x660E;&#x3092;&#x63D0;&#x4F9B;&#x3059;&#x308B;&#x306B;&#x306F;&#x307E;&#x305F;&#x3001;&#x30DE;&#x30F3;&#x30AC;&#x30F3;&#x518D;&#x69CB;&#x6210;&#x9175;&#x7D20;&#x89E6;&#x5A92;&#x975E;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6; E101N/D266N &#x5909;&#x7570;&#x4F53;&#x306E;&#x57FA;&#x8CEA;&#x7D50;&#x5408;&#x69CB;&#x9020;&#x306E;&#x89E3;&#x50CF;&#x5EA6; 2.1 &#x306B;&#x5F97;&#x3089;&#x308C;&#x305F; &Aring;&#x3002;&#x914D;&#x4F4D;&#x5B50;, &#x30DE;&#x30F3;&#x30AC;&#x30F3;&#x518D;&#x69CB;&#x6210; E101N &#x5909;&#x7570;&#x89E3;&#x50CF;&#x5EA6; 2.6 &Aring;&#x3002;&#x914D;&#x4F4D;&#x5B50;&#x7121;&#x6599;&#x4E9C;&#x925B;&#x518D;&#x69CB;&#x6210;&#x91CE;&#x751F;&#x578B;&#x306E;&#x69CB;&#x9020;&#x3001;E101N&#x3001;R230D&#x3001;&#x304A;&#x3088;&#x3073; E101G/R230C &#x5909;&#x7570;&#x682A; GKL &#x306E; 2.1 &#x306E;&#x89E3;&#x50CF;&#x5EA6;&#x306B; &Aring;&#x3001;2.1 &Aring;&#x3001;1.9 &Aring;&#x3001;&#x304A;&#x3088;&#x3073; 2.0 &Aring;&#x3001;&#x305D;&#x308C;&#x305E;&#x308C;&#x3002;&#x7279;&#x306B;&#x3001;GKL &#x9032;&#x5316;&#x3057;&#x305F; E101G/R230C &#x5909;&#x7570;&#x4F53;&#x306E;&#x69CB;&#x9020;&#x306F;&#x89E6;&#x5A92;&#x4F5C;&#x7528;&#x306E;&#x89B3;&#x6E2C;&#x306E;&#x5411;&#x4E0A;&#x306B;&#x8CA2;&#x732E;&#x3059;&#x308B;&#x89E6;&#x5A92;&#x751F;&#x7523;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6; &#x30B5;&#x30A4;&#x30C8; &#x30A2;&#x30FC;&#x30AD;&#x30C6;&#x30AF;&#x30C1;&#x30E3;&#x306E;&#x305F;&#x3081;&#x306E;&#x8A3C;&#x62E0;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x307E;&#x3059;&#x3002;&#x9AD8;&#x6FC3;&#x5EA6;&#x3067;&#x3001;&#x91CE;&#x751F;&#x578B;&#x3068;&#x5909;&#x7570;&#x578B;&#x306E; GKL &#x9175;&#x7D20;&#x7279;&#x7570;&#x7684; 512 &#x306E;&#x7BC4;&#x56F2;&#x3067;&#x5438;&#x5149;&#x5EA6;&#x306E;&#x6700;&#x5927;&#x5024;&#x3092;&#x7740;&#x8272;&#x3055;&#x308C;&#x3066;&#x3042;&#x308A;&#x3001;553 nm nm&#x3002;&#x91CE;&#x751F;&#x578B;&#x3068;&#x5909;&#x7570;&#x578B; GKL &#x69CB;&#x9020;&#x7740;&#x8272;&#x306E;&#x8D77;&#x6E90;&#x3068; &alpha;-&#x967D;&#x30A4;&#x30AA;&#x30F3;&#x3068; Tyr99 &#x306E;&#x9593;&#x96FB;&#x8377;&#x79FB;&#x52D5;&#x932F;&#x4F53;&#x4E2D;&#x306E;&#x9175;&#x7D20;&#x306E;&#x30B5;&#x30A4;&#x30C8;&#x5185;&#x306B;&#x6271;&#x3044;&#x3084;&#x3059;&#x3044;&#x30EA;&#x30F3;&#x30AF;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x307E;&#x3059;&#x3002;&#x4E00;&#x7DD2;&#x306B;&#x53D6;&#x3089;&#x308C;&#x3066;&#x3001;&#x3053;&#x306E;&#x7814;&#x7A76;&#x306F;&#x9175;&#x7D20;&#x306E;&#x89E6;&#x5A92;&#x9175;&#x7D20;&#x6D3B;&#x6027;&#x90E8;&#x4F4D;&#x306E;&#x69CB;&#x9020;&#x306E;&#x5FAE;&#x5999;&#x306A;&#x5909;&#x5316;&#x3092; modulability &#x306E;&#x305F;&#x3081;&#x306E;&#x8A3C;&#x62E0;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x307E;&#x3059;&#x3002;

&#x751F;&#x7523;&#x6027;&#x306E;&#x9AD8;&#x3044;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6; &#x30B5;&#x30A4;&#x30C8; &#x30A2;&#x30FC;&#x30AD;&#x30C6;&#x30AF;&#x30C1;&#x30E3;&#x306F;&#x9032;&#x5316;&#x3057;&#x305F;&#x30AF;&#x30A9;&#x30FC;&#x30E9;&#x30E0;&#x6D88;&#x708E; GKL Lactonase &#x306E;&#x69CB;&#x9020;&#x306E;&#x8A3C;&#x62E0;&#x3067;&#x306F;&#x3002;

&#xD5A5;&#xC0C1; &#xB41C; reactivities&#xC640; lactonases &#xCFFC;&#xB7FC; &#xB2F4;&#xAE08;&#xC9C8;&#xC758; &#xACF5;&#xD559;&#xACFC; &#xC0DD;&#xCCB4; &#xC678;&#xC5D0;&#xC11C; &#xC9C4;&#xD654; thermostable GKL &#xD6A8;&#xC18C; &#xCD09;&#xB9E4; &#xD65C;&#xB3D9; &#xC99D;&#xAC00; &#xBC0F; &#xD655;&#xB300; &#xAE30;&#xD310; &#xBC94;&#xC704; E101G/R230C GKL &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xC0DD;&#xC131; &#xD15C;&#xD50C;&#xB9BF;&#xC73C;&#xB85C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xB2EC;&#xC131; &#xD588;&#xB2E4; [&#xC6B0;, J. Y., Xue, B., &#xB9AC;, K. H., &#xD241; &#xCCAD;, A., &#xC6B0;, L., &#xB85C;&#xBE48;&#xC2A8;, R. C. &#xBC0F; &#xC720;, W. S. (2010) J Biol &#xCF10; 285, 40911-40920]. &#xC774; &#xD6A8;&#xC18C; (&beta;/&alpha;) 8-&#xCD1D; &#xC2E0; &#xBC30;&#xB97C; &#xC18C;&#xC720; &#xD558; &#xACE0; PLL (Phosphotriesterase-Like Lactonase)&#xC758; &#xD68C;&#xC6D0;&#xC785;&#xB2C8;&#xB2E4;-hydrolyze &#xBC15;&#xD14C;&#xB9AC;&#xC544;&#xC758; &#xCFFC;&#xB7FC; &#xC13C; &#xC2F1; &#xD1B5;&#xB85C; &#xC911;&#xC7AC; N acyl homoserine lactones amidohydrolase superfamily &#xB0B4;&#xC5D0;&#xC11C; &#xD6A8;&#xC18C;&#xC758; &#xADF8;&#xB8F9;. &#xC9C4;&#xD654; N butyryl L homoserine lactone (&#xAE30;&#xD310;)&#xC758; &#xAD6C;&#xC870;-2.2&#xC758; &#xD574;&#xC0C1;&#xB3C4;&#xB97C; inactivating D266N &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xC874;&#xC7AC; &#xBC14;&#xC6B4;&#xB4DC; E101G/R230C GKL &#xD6A8;&#xC18C; &#xD574;&#xACB0; &#xB418;&#xC5C8;&#xB2E4; &Aring; &#xAD00;&#xCC30; &#xB41C; &#xC18D;&#xB3C4; &#xD5A5;&#xC0C1;;&#xC5D0; &#xB300; &#xD55C; &#xC124;&#xBA85;&#xC744; &#xC81C;&#xACF5; &#xD558;&#xAE30; &#xC704;&#xD574; &#xB610;&#xD55C;, &#xB9DD;&#xAC04; &#xC7AC;&#xAD6C;&#xC131; &#xD6A8;&#xC18C;&#xC758; catalytically &#xBE44;&#xD65C;&#xC131; E101N/D266N &#xB3CC;&#xC5F0;&#xBCC0;&#xC774;&#xC758; &#xAE30;&#xD310; &#xBC14;&#xC778;&#xB529; &#xAD6C;&#xC870; 2.1 &#xD574;&#xC0C1;&#xB3C4;&#xB85C; &#xC5BB;&#xC740; &Aring;; 2.6&#xC758; &#xD574;&#xC0C1;&#xB3C4;&#xB97C; &#xB9AC;&#xAC04;&#xB4DC; &#xBB34;&#xB8CC;, &#xB9DD;&#xAC04; &#xC7AC;&#xAD6C;&#xC131; E101N &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &Aring;; &#xADF8;&#xB9AC;&#xACE0; &#xB9AC;&#xAC04;&#xB4DC; &#xBB34;&#xB8CC; &#xC544;&#xC5F0; &#xC7AC;&#xAD6C;&#xC131; &#xC57C;&#xC0DD;-&#xD0C0;&#xC785;&#xC758; &#xAD6C;&#xC870;, GKL&#xC758; 2.1&#xC758; &#xD574;&#xC0C1;&#xB3C4; E101N, R230D, &#xBC0F; E101G/R230C &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &Aring;, 2.1 &Aring;, 1.9 &Aring;, &#xBC0F; 2.0 &Aring;, &#xAC01;&#xAC01;. &#xD2B9;&#xD788;, GKL&#xC758; &#xC9C4;&#xD654; E101G/R230C &#xB3CC;&#xC5F0;&#xBCC0;&#xC774;&#xC758; &#xAD6C;&#xC870; &#xCD09;&#xB9E4;&#xC5D0;&#xC11C; &#xAD00;&#xCC30; &#xB41C; &#xD5A5;&#xC0C1;&#xC5D0; &#xAE30;&#xC5EC; &#xD558;&#xB294; catalytically &#xC0DD;&#xC0B0;&#xC131; &#xC561;&#xD2F0;&#xBE0C; &#xC0AC;&#xC774;&#xD2B8; &#xC544;&#xD0A4;&#xD14D;&#xCC98;&#xC5D0; &#xB300; &#xD55C; &#xC99D;&#xAC70;&#xB97C; &#xC81C;&#xACF5; &#xD569;&#xB2C8;&#xB2E4;. &#xB192;&#xC740; &#xB18D;&#xB3C4;&#xC5D0;&#xC11C; &#xC57C;&#xC0DD; &#xD615; &#xBC0F; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; GKL &#xD6A8;&#xC18C;&#xB294; &#xCC28;&#xB3D9; &#xC0C9;&#xAE54;, 512&#xC758; &#xBC94;&#xC704;&#xC5D0;&#xC11C; &#xD761; &#xAD11;&#xB3C4; &#xCD5C;&#xB300;&#xAC12;&#xC73C;&#xB85C; nm 553 ~ nm. &#xC57C;&#xC0DD; &#xD615; &#xBC0F; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; GKL&#xC758; &#xAD6C;&#xC870; &#xC0AC;&#xC774;&#xB294; &#xCC44;&#xC0C9;&#xC758; &#xAE30;&#xC6D0; &#xBC0F; &#xD6A8;&#xC18C; &#xD65C;&#xC131; &#xC0AC;&#xC774;&#xD2B8; &#xB0B4;&#xC5D0;&#xC11C; &alpha;-&#xC591;&#xC774;&#xC628;&#xACFC; Tyr99 &#xC0AC;&#xC774; &#xCDA9;&#xC804; &#xC804;&#xC1A1; &#xB2E8;&#xC9C0; &#xC628;&#xC21C;&#xD55C; &#xB9C1;&#xD06C;&#xB97C; &#xC81C;&#xACF5;&#xD569;&#xB2C8;&#xB2E4;. &#xD568;&#xAED8; &#xCC0D;&#xC740;&#xC774; &#xC5F0;&#xAD6C;&#xB294; &#xD6A8;&#xC18C; &#xCD09;&#xB9E4; &#xD6A8;&#xC18C; &#xD65C;&#xC131; &#xC0AC;&#xC774;&#xD2B8; &#xC544;&#xD0A4;&#xD14D;&#xCC98;&#xC5D0; &#xBBF8;&#xBB18;&#xD55C; &#xBCC0;&#xD654;&#xB97C; &#xD1B5;&#xD574; modulability&#xC5D0; &#xB300; &#xD55C; &#xC99D;&#xAC70;&#xB97C; &#xC81C;&#xACF5; &#xD569;&#xB2C8;&#xB2E4;.

&#xC9C4;&#xD654; &#xCFFC;&#xB7FC; &#xD01C;&#xCE6D; GKL Lactonase&#xC5D0; &#xB300; &#xD55C; &#xC0DD;&#xC0B0;&#xC801;&#xC778; &#xC561;&#xD2F0;&#xBE0C; &#xC0AC;&#xC774;&#xD2B8; &#xC544;&#xD0A4;&#xD14D;&#xCC98;&#xC758; &#xAD6C;&#xC870;&#xC801; &#xC99D;&#xAC70;.

La evoluci&oacute;n in vitro y la ingenier&iacute;a de lactonases apagando el qu&oacute;rum con mayor reactividades logr&oacute; utilizando una enzima GKL termoestable como plantilla, produciendo el mutante GKL E101G/R230C con aumento de la actividad catal&iacute;tico y sustrato ampliada gama [Chow, J. Y., Xue, B., Lee, K. H., Tung, A., Wu, L., Robinson, C. R. y Tejo, W. S. (2010) J Biol Chem 285, 40911-40920]. Esta enzima posee el doble de 8 barriles (&alpha;/&beta;) y es miembro del PLL (Phosphotriesterase-Like Lactonase)-grupo de enzimas dentro de la Superfamilia amidohidrolasa que hidrolizan N-acil-homoserina lactonas, que median las v&iacute;as quorum-sensing de bacterias. La estructura de la evolucionada N-Butiril-L-Homoserina lactona (substrato)-enzima de E101G/R230C GKL consolidado se resolvi&oacute;, en presencia de la mutaci&oacute;n D266N inactivador, a una resoluci&oacute;n de 2.2 &Aring; para proporcionar una explicaci&oacute;n para las mejoras de la tasa observada; Adem&aacute;s, la estructura del sustrato-limite del mutante E101N/D266N catal&iacute;tico inactiva de la enzima manganeso-reconstituido se obtuvo a una resoluci&oacute;n de 2.1 &Aring;; el mutante de E101N libre de ligando, manganeso-reconstituido a una resoluci&oacute;n de 2.6 &Aring;; y las estructuras del ligando libre cinc-reconstituido-tipo salvaje, los mutantes E101N, R230D y E101G/R230C de GKL a una resoluci&oacute;n de 2.1 &Aring;, 2.1 &Aring;, 1,9 &Aring; y 2.0 &Aring;, respectivamente. En particular, la estructura del mutante E101G/R230C evolucionada de GKL proporciona la evidencia para una arquitectura activa del sitio catal&iacute;tico productiva que contribuye a la mejora observada en cat&aacute;lisis. En altas concentraciones, enzimas de tipo salvaje y mutantes GKL son diferencialmente coloreadas, con m&aacute;ximos de absorbancia en el rango de 512 nm a 553 nm. Las estructuras de la GKL tipo salvaje y mutante proporcionan un enlace manejable entre los or&iacute;genes de la coloraci&oacute;n y el complejo de transferencia de carga entre el &alpha;-caci&oacute;n y Tyr99 en el sitio de activo de la enzima. Tomados en conjunto, este estudio proporciona evidencia para la modulable de cat&aacute;lisis enzim&aacute;tica a trav&eacute;s de cambios sutiles en la arquitectura del sitio activo de enzima.

Pruebas estructurales de una arquitectura de activos productivos del sitio para una evolucionada qu&oacute;rum-amortiguamiento GKL Lactonase.

Acinetobacter baumannii is a major human pathogen associated with multidrug-resistant nosocomial infections; its virulence is attributed to quorum-sensing-mediated biofilm formation, and disruption of biofilm formation is an attractive antivirulence strategy. Here, we report the first successful demonstration of biofilm disruption in a clinical isolate of A. baumannii S1, using a quorum-quenching lactonase obtained by directed evolution; this engineered lactonase significantly reduced the biomass of A. baumannii-associated biofilms, demonstrating the utility of this antivirulence strategy.

Disruption of biofilm formation by the human pathogen Acinetobacter baumannii using engineered quorum-quenching lactonases.

Terpenoid synthases construct the carbon skeletons of tens of thousands of natural products. To predict functions and specificity of triterpenoid synthases, a mechanism-based, multi-intermediate docking approach is proposed. In addition to enzyme function prediction, other potential applications of the current approach, such as enzyme mechanistic studies and enzyme redesign by mutagenesis, are discussed.

Predicting the functions and specificity of triterpenoid synthases: a mechanism-based multi-intermediate docking approach.

Terpenoids are a large structurally diverse group of natural products with an array of functions in their hosts. The large amount of genomic information from recent sequencing efforts provides opportunities and challenges for the functional assignment of terpene synthases that construct the carbon skeletons of these compounds. Inferring function from the sequence and/or structure of these enzymes is not trivial because of the large number of possible reaction channels and products. We tackle this problem by developing an algorithm to enumerate possible carbocations derived from the farnesyl cation, the first reactive intermediate of the substrate, and evaluating their steric and electrostatic compatibility with the active site. The homology model of a putative pentalenene synthase (Uniprot: B5GLM7) from Streptomyces clavuligerus was used in an automated computational workflow for product prediction. Surprisingly, the workflow predicted a linear triquinane scaffold as the top product skeleton for B5GLM7. Biochemical characterization of B5GLM7 reveals the major product as (5S,7S,10R,11S)-cucumene, a sesquiterpene with a linear triquinane scaffold. To our knowledge, this is the first documentation of a terpene synthase involved in the synthesis of a linear triquinane. The success of our prediction for B5GLM7 suggests that this approach can be used to facilitate the functional assignment of novel terpene synthases.

Jeng Yeong Chow

Department of Chemistry

University of Utah

Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases