Mechanism and Specificity of an Acyltransferase Domain from a Modular Polyketide Synthase

Dunn, Briana J.; Cane, David E.; Khosla, Chaitan

doi:10.1021/bi400185v

Cited by 63 publications

(150 citation statements)

References 16 publications

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“…Recently, the AT domain from module 3 of DEBS was characterized using a coupled enzymatic assay that allowed for continuous monitoring of AT catalytic activity and thus characterization of a-carboxyacyl-CoA and ACP specificity. This study revealed that the (2S)-methylmalonyl-CoA specificity of this enzyme lies in the first half reaction of the ping-pong mechanism (formation of the methylmalonyl-AT intermediate; [39]). Hydrolytic cleavage of incorrect substrates has been hypothesized to be a specificity-determining mechanism used by AT domains, but the reported importance of this mechanism has varied [39,41].…”

Section: Acyltransferase Mechanistic Insightsmentioning

confidence: 86%

“…The relative kinetic parameters describing the specificity of a mutated AT domain cannot be determined using in vivo techniques, and until recently the kinetic effects of such mutations were not known. In vitro analysis of a YASH to HAFH DEBS AT3 mutant, along with another point mutant previously examined in vivo [58,60] revealed that the activity of these mutants is drastically attenuated, implying that incorporation of non-natural extender units likely occurs as a result of diminished AT catalytic activity and not because of increased specificity towards the nonnative substrate [39]. The engineering of AT domains by site-directed mutagenesis therefore has vast room for improvement and will benefit greatly from the use of advanced sequence-and structure-based techniques.…”

Section: Acyltransferase Site-directed Mutagenesismentioning

confidence: 93%

“…AT-catalysed incorporation of CoA-derived extender units occurs by a ping-pong bi-bi mechanism [38,39] involving an acyl-AT intermediate that is subject to nucleophilic attack by the thiol residue found on the phosphopantetheine arm of the ACP. Early studies revealed that modular AT domains likely do not possess epimerase activity, and all domains in DEBS exclusively incorporate (2S)-methylmalonyl-CoA [40].…”

Section: Acyltransferase Mechanistic Insightsmentioning

confidence: 99%

See 2 more Smart Citations

Engineering the acyltransferase substrate specificity of assembly line polyketide synthases

Dunn

Khosla

2013

J. R. Soc. Interface.

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107

114

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Polyketide natural products act as a broad range of therapeutics, including antibiotics, immunosuppressants and anti-cancer agents. This therapeutic diversity stems from the structural diversity of these small molecules, many of which are produced in an assembly line manner by modular polyketide synthases. The acyltransferase (AT) domains of these megasynthases are responsible for selection and incorporation of simple monomeric building blocks, and are thus responsible for a large amount of the resulting polyketide structural diversity. The substrate specificity of these domains is often targeted for engineering in the generation of novel, therapeutically active natural products. This review outlines recent developments that can be used in the successful engineering of these domains, including AT sequence and structural data, mechanistic insights and the production of a diverse pool of extender units. It also provides an overview of previous AT domain engineering attempts, and concludes with proposed engineering approaches that take advantage of current knowledge. These approaches may lead to successful production of biologically active 'unnatural' natural products.

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Section: Acyltransferase Mechanistic Insightsmentioning

confidence: 86%

Section: Acyltransferase Site-directed Mutagenesismentioning

confidence: 93%

Section: Acyltransferase Mechanistic Insightsmentioning

confidence: 99%

See 1 more Smart Citation

Engineering the acyltransferase substrate specificity of assembly line polyketide synthases

Dunn

Khosla

2013

J. R. Soc. Interface.

Self Cite

107

114

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“…Sin embargo, faltó purificar mayor cantidad de las fracciones para poder seguir haciendo estudios de cristalografía y secuenciamiento, con lo que podríamos confirmar que la Fracción A corresponde a la papaína. Finalmente, dado que la caracterización de las proteasas está vinculada a su rol como catalizador biológico, se debería estudiar la forma que adopta la proteína, específica-mente su sitio activo, ya que ello permitiría conocer más acerca de su función y sus implicaciones en los mecanismos reguladores de la vida; esto se puede obtener primero cristalizando la proteína (Dunn et al, 2013). Además, se deberían estudiar estas proteasas mediante las técnicas de "ionización suave" (MALDI y electrospray) que, en combinación con diversas técnicas de análisis (espectrometría de masas), nos permitan identificar y secuenciar las proteasas aisladas (Obregón et al, 2007a;2007b), lo cual podría confirmar que la Fracción A es la papaína de "Mito".…”

Section: Conclusionesunclassified

Purification and preliminary characterization of latex proteases of Vasconcellea candicans (A. Gray) A. DC (Mito)

Gutiérrez¹,

Nolasco²,

Cruz³

2017

sci. agropecu

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ResumenEstudios preliminares indican que, el látex fresco del "Mito" tiene una actividad específica de papaína de 1,84 veces mayor a la encontrada en el látex of papaya, por lo que el objetivo de este trabajo fue purificar y caracterizar las proteasas del látex fresco del "Mito" que tuvieran actividad de papaína. El extracto crudo de proteasas se obtuvo a partir del látex de "Mito" el cual fue resuspendido (1:1) en buffer acetato de Na 10 mM a pH 5,0; inmediatamente se precipitaron proteínas a pH 9,0 y luego con sulfato de amonio al 45%. Posteriormente se purificó en una columna de Sephadex G-100 y se obtuvieron tres fracciones: A, B y C; utilizando como sustrato caseína se midió la actividad enzimática específica (AEE). Se encontró que para la Fracción A la AEE fue de 87,74 nkat.mg -1 proteína, para la Fracción B fue de 14,93 nkat.mg -1 proteína y para la Fracción C fue de 16,13 nkat.mg -1 proteína. La AEE de la fracción A frente a la de papaína de látex fresco de C. papaya fue 13,3 veces mayor. En el análisis electroforético (gel desnaturalizante, 12%) se observa para la fracción A dos bandas de proteínas teniendo una de ellas una "relación de frente" semejante al estándar de papaína. Además, se observó que la fracción A (papaína de "Mito") frente a diferentes concentraciones de caseína, usada como sustrato, presenta una curva sigmoidea michaeliana; a diferentes volúmenes de enzima se muestra un comportamiento lineal; tiene un pH óptimo a 7,5 y es activa hasta 60 ºC.Palabras clave: Lomas de Perú; papaya silvestre; látex; papaína; Vasconcella candicans (A. Gray). AbstractPreliminary studies indicate that, the "Mito" fresh latex, has a specific activity of papain from 1.84 times greater than that found in the latex of papaya, so the objective of this study was to purify and characterize "Mito" fresh latex proteases that have activity of papain. The crude extract protease was obtained from the "Mito" latex which was re-suspended (1:1) in 10 mM Na acetate buffer at pH 5.0; immediately proteins were precipitated at pH 9.0 and then with 45% ammonium sulfate. Subsequently, the proteins were purified on a Sephadex G-100 column and were three fractions: A, B and C. Using as a substrate casein, the enzymatic specific activity (ESA) was measured and was found to be the fraction A was 87.74 nkat.mg -1 protein, for fraction B was 14.93 nkat.mg -1 protein and for fraction C it was 16.13 nkat.mg -1 protein. ESA of fraction A against papain of fresh latex of C. papaya was 13.3 times greater. Electrophoretic analysis (12% denaturant gel) shows for A fraction, two protein bands having one of them a relation similar to the papain standard. In addition, there was observed that the A fraction (papain of "Mito") against different concentrations of casein, used as a substrate, displays a michaeliane sigmoid curve; different volumes of enzyme shows a linear behavior; it has an optimum pH of 7.5 and is active up to 60 °C.

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“…[21,22] Skiniotis and co-workers have presented a new model of PKSmediated natural compound synthesis based on moderate-resolution PikAIII data and biochemical studies. The new insight will change our view of PKSs.…”

Section: Integrity Of the Ks-at Unitmentioning

confidence: 99%

Modular Polyketide Synthases (PKSs): A New Model Fits All?

Rittner¹,

Grininger²

2014

ChemBioChem

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Mechanism and Specificity of an Acyltransferase Domain from a Modular Polyketide Synthase

Cited by 63 publications

References 16 publications

Engineering the acyltransferase substrate specificity of assembly line polyketide synthases

Engineering the acyltransferase substrate specificity of assembly line polyketide synthases

Purification and preliminary characterization of latex proteases of Vasconcellea candicans (A. Gray) A. DC (Mito)

Modular Polyketide Synthases (PKSs): A New Model Fits All?

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