Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Reexamination Certificate
2001-01-05
2002-11-05
Leary, Louise N. (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
C435S007500, C435S007100, C435S975000, C435S968000, C435S004000
Reexamination Certificate
active
06475751
ABSTRACT:
FIELD OF THE INVENTION
This invention pertains generally to biological assays of components of a fatty acid synthase, and more particularly, to the use of scintillation proximity assays suitable for detecting and measuring activities such as that of &bgr;-ketoacyl-acyl carrier protein synthase III.
BACKGROUND OF THE INVENTION
The pathway for the biosynthesis of saturated fatty acids is very similar in prokaryotes and eukaryotes. However, while the chemical reactions may not vary, the organization of the biosynthetic apparatus is very different. Vertebrates and yeast possess type I fatty acid synthases (FASs) in which all of the enzymatic activities of the fatty acid biosynthetic pathway are encoded on one or two polypeptide chains, respectively, and an acyl carrier protein (ACP) is an integral part of the complex. In contrast, in most bacterial and plant type II FASs, each of the reactions is catalyzed by distinct monofunctional enzymes and the ACP is a discrete protein. Mycobacteria are unique in that they possess both type I and II FASs; the former is involved in basic fatty acid biosynthesis whereas the latter is involved in synthesis of complex cell envelope lipids such as mycolic acids.
The fatty acid biosynthetic process is divided into three phases: initiation, elongation and termination. The elongation phase is cyclical, in that acyl thioester (typically attached to an acyl carrier protein) is repeatedly elongated two carbons at a time using malonyl CoA as the 2-carbon “extender” unit.
In type II fatty acid biosynthesis (refer to
FIG. 1
) the first elongation step or initiation step is the condensation of malonyl-ACP (MACP) with an acyl-CoA which is catalyzed by &bgr;-ketoacyl-acyl carrier protein synthase III (KAS III or FabH, see definitions). The product of this reaction is &bgr;-ketoacyl ACP which then enters the reduction component of the cycle by undergoing ketoester reduction by NADPH-dependent &bgr;-ketoacyl-ACP reductase (FabG, “KR” in FIG.
1
). Subsequent dehydration by &bgr;-hydroxyacyl-ACP dehydrase (either FabA or FabZ, represented by “DH” in
FIG. 1
) leads to trans-2-enoyl-ACP which is in turn converted to acyl-ACP by NADH-dependent enoyl-ACP reductase (FabI, “ER” in FIG.
1
). Synthases KAS I and KAS II (also known as FabB and FabF, see definitions) then catalyze a 2-carbon elongation of acyl-ACP via condensation with MACP, followed by ketoester reduction, dehydration, and enoyl reduction as described above. Further rounds of this cycle, adding two carbon atoms per cycle, eventually lead to production of a fatty acid of the desired length, whereupon the cycle is stopped largely due to feedback inhibition of KASIII (Heath, et al, (1996),
J.Biol.Chem.
271, 1833-1836). KASIII is therefore a major biosynthetic enzyme which is also a key regulatory point in the overall biosynthetic pathway (Heath, R. J. and Rock, C. O. 1996.
J.Biol.Chem.
271, 1833-1836; Heath, R. J. and Rock, C. O. 1996.
J.Biol.Chem.
271, 10996-11000). KASIII from different organisms exhibit different acyl CoA specificity, which appears to dictate the type of fatty acid product made. Thus,
E. coli
makes a mixture of odd and even-number straight chain fatty acids and its KASIII reacts preferentially with acetyl CoA and propionyl CoA (Heath and Rock, 1996.
J.Biol.Chem.
271, 10996-11000).
Streptomyces glauecescens
and
Bacillus subtilis
make both branched and straight-chain fatty acids. The KASIII from these organisms have been shown to have a more relaxed substrate specificity, reacting with substrates such as isobutyryl CoA as well acetyl CoA and propionyl CoA (Choi, K. H., Heath, R. J. and Rock, C. O. 2000. &bgr;-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis.
J Bacteriol
182: 365-70; Han, L., Lobo, S. and Reynolds, K. A. 1998. Characterization of 3-ketoacyl acyl carrier protein synthase III from
Streptomyces glaucescens
: Its role in the initiation of fatty acid biosynthesis.
J Bacteriol
180: 4481-4486). The KASIII of Mycobacterium tuberculosis on the other hand appears to have a substrate preference for long chain acyl CoA substrates and appears to be responsible for initiation of meromycolate fatty acid biosynthesis (Choi, K. H., Kremer, L., Besra, G. S., and Rock, C. O. 2000.
J. Biol. Chem.
)
The enzymes involved in type II fatty acid biosynthesis represent attractive targets for modulation of fatty acid synthesis in prokaryotes and plants. Substances that modulate type II fatty acid synthesis could potentially function as therapeutic agents (for example, as antibiotics), or as herbicides. Typically, the screening of potential enzymic effectors is carried out via high throughput screening (HTS) techniques. However, the currently available assay procedures for type II FAS enzymes are inadequate for HTS. For example, the traditional measurement of KASIII activity is a coupled enzyme trichloroacetic acid (TCA) precipitation assay (Han, L. et al. 1998
J.Bacteriol.
180, 4481-4486). In this assay, malonyl CoA-ACP transacylase (FabD, “MAT” in
FIG. 1
) is utilized to convert malonyl-CoA to malonyl-ACP. KAS III then catalyzes the condensation of malonyl-ACP with acetyl-CoA which has been radiolabeled, resulting in the production of the radiolabeled product 3-ketoacyl-ACP. An acid precipitation step is then used to physically separate the radioactive 3-ketoacyl-ACP product from the radiolabeled acetyl-CoA substrate. In addition to the precipitation step, an acid-washing step is also required to remove any residual radiolabeled acetyl-CoA from the precipitate. Only after these steps are completed can the radiolabeled, precipitated product be quantitated by scintillation counting.
A variation of this assay method involves binding the 3-ketoacyl ACP product to filter paper disks (such as Whatmann 3MM). These disks are then washed with three changes of ice cold tricchloracteric acid and the filters dried and counted using scintillation cocktail (Choi, Heath and Rock, 2000
. J Bacteriol.
182, 365-370). Finally, a gel electrophoresis method has been described. This involves the use of radiolabeled malonyl CoA which is converted in the assay to malonyl ACP by the action of FabD. KASIII then catalyzes the formation of a radiolabeled ketoacyl ACP product using this malonyl ACP and a non-radioactive acyl CoA substrate. The radiolabeled product to reduced to the corresponding 3-hydroxyacyl ACP product by the action of FabG and NADPH and is then resolved from the other components of the assay by use of a polyacrylamide gel. Product quantitation is then obtained by exposure of the gel to a PhosphoImager screen (Choi, Heath and Rock, 2000.
J Bacteriol.
182, 365-370).
Obviously, such multistep assay procedures are ill suited for high throughput screening methods. It would be highly desirable to have available a faster and more direct assay for the activities of the enzymes involved in type II fatty acid biosynthesis in order to screen libraries of their potential effectors at an acceptable rate and in a cost-effective manner.
SUMMARY OF THE INVENTION
The bacterial fatty acid biosynthesis pathway is a selective target for the development of novel antibiotics for treating infectious disease, (as shown in U.S. Pat. No. 5,614,551 which is incorporated herein by this reference). This selectivity is based both on the significant differences in the fatty acid synthases (FASs) of prokaryotes and eukaryotes and on their relative physiological importance. In higher organisms such as mammals, a multifunctional enzyme complex Type I fatty acid synthase (FAS) in which all of the enzymatic activities are encoded on one or two polypeptide chains, catalyzes the biosynthetic pathway. In humans, this pathway under most conditions is down-regulated due to exogenous dietary lipid intake. In contrast, fatty acid biosynthesis in bacteria appears to be an essential process catalyzed by a set of dissociable enzymes known collectively as a Type II FAS. The natural product thiolactomycin, a thiolactone antibiotic with in vitro and in vivo act
He Xin
Reynolds Kevin A.
Leary Louise N.
Virginia Commonwealth University
Whitham Curtis & Christofferson, P.C.
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