Glyoxysomal acetoacetyl-CoA thiolase and 3-oxoacyl-CoA thiolase from sunflower cotyledons

Abstract

Following chromatography on hydroxyapatite, the elution profile of the thiolase activity of the glyoxysomal fraction from sunflower (Helianthus annuus L.) cotyledons exhibited two peaks when the enzyme activity was assayed with acetoacetyl-CoA as substrate. Only one of these two activity peaks was detectable when a long-chain thiolase substrate was used in the activity assay. The proteins (thiolase I and thiolase II) underlying the two activity peaks detected with acetoacetyl-CoA were of glyoxysomal origin. They were purified using glyoxysomal matrices as starting material, and biochemically characterized. Thiolase I is an acetoacetyl-CoA thiolase (EC 2.3.1.9) exhibiting activity only towards acetoacetyl-CoA (Km = 11 microM). Its contribution to the total glyoxysomal thiolytic activity towards acetoacetyl-CoA amounted to about 15%. Thiolase II is a 3-oxoacyl-CoA thiolase (EC 2.3.1.16). The activity of the enzyme towards 3-oxoacyl-CoAs increased with increasing chain length of the substrate. Thiolase II exhibited a Km value of 27 microM with acetoacetyl-CoA as substrate. and Km values between 3 and 7 microM with substrates having a carbon chain length from 6 to 16 carbon atoms. The thiolase activity of the glyoxysomes towards acetoacetyl-CoA and 3-oxopalmitoyl-CoA exceeded the glyoxysomal butyryl-CoA and palmitoyl-CoA beta-oxidation rates, respectively, by about 10-fold at all substrate concentrations employed (1-15 microM).

Cloning, Expression and Purification of an Acetoacetyl CoA Thiolase from Sunflower Cotyledon

“…At a concentration of 100 mM imidazole, the recombinant thiolase is also present but is more significantly contaminated with higher and lower molecular weight proteins (Figure 5, lane 6). The size of the AACT purified and characterized from the glyoxysomal fraction of sunflower cotyledons was reported as 63 kDa 13, which is significantly higher than the molecular weight reported here; however, another thiolase activity was reported in that paper and was not further characterized. This second thiolase perhaps corresponds to the thiolase reported here.…”

“…Extensive characterization of two thiolase enzymes from the glyoxysomal fraction of sunflower cotyledon tissue has shown that both the Thiolase I and Thiolase II activities occur in this tissue 13. We have previously cloned and purified an enzymatically active 3-ketoacyl CoA thiolase from sunflower cotyledon 14, and here we report the cloning, expression and purification of the acetoacetyl CoA thiolase from the sunflower cotyledon.…”

The Crystal Structure of a Plant 3-Ketoacyl-CoA Thiolase Reveals the Potential for Redox Control of Peroxisomal Fatty Acid β-Oxidation

“…It is here that the control of storage fat degradation and channelling of the products towards the biosynthesis of essential compounds such as carbohydrates occurs. [11][12][13][14][15] Peroxisomal KAT is produced in vivo as a precursor carrying a cleavable N-terminal targeting signal. 16,17 The N-terminal segment is not required for activity, nor does it influence the quaternary structure, which is homodimeric in the mature conformation.…”

Función del gen 3-cetoacil-CoA tiolasa 2 (KAT2) en defensa y desarrollo. Participación de los peroxisomas en las respuestas a herida en Arabidopsis thaliana.

“…En otras especies vegetales como pepino, también se han caracterizado distintas isoenzimas MFP expresadas diferencialmente en distintos tejidos de la planta y con distintas actividades en cada caso (Behrends et al, 1988;Preisig-Muller et al, 1994; Guhnemann-Schafer y Kindl, 1995a;. Por último, las proteínas MFP presentan varias regiones de unión a microtúbulos que le permiten el transporte a través de los mismos, e implica que estas proteínas se encuentren asociadas a microtúbulos además de su localización peroxisomal (Chuong et al, 2002; Kindl, 1993), mango (Bojorquez y Gómez-Lim, 1995), colza (Olesen y Brandt, 1995), cotiledones de calabaza (Kato et al, 1996), girasol (Oeljeklaus et al, 2002) y en Arabidopsis (Hayashi et al, 1998b;Germain et al, 2001). El genoma de Arabidopsis contiene tres genes que codifican proteínas KAT.…”

“…En los últimos años, se ha confirmado la presencia de actividad enzimática KAT en varias especies (Olesen et al, 1997;Germain et al, 2001;Oeljeklaus et al, 2002) y ha sido obtenida la estructura cristalina de la enzima de Arabidopsis (Sundaramoorthy et al, 2006).…”