Enoyl reductases and methods of use thereof

Details Enoyl reductases and methods of use thereof Enoyl reductases and methods of use thereof

2000-02-04

2003-09-02

Duffy, Patricia A. (Department: 1645)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Oxidoreductase

C435S183000

Reexamination Certificate

active

06613553

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to novel enzymes that act as enoyl reductases. Two distinct families of enoyl reductases have been identified in bacteria, each of which have a consensus amino acid sequence. The enoyl reductases can be used as targets for designing both new prophylactics and treatments for bacterial infections. Nucleic acid and amino acid sequences of the novel enoyl reductases are also provided.
BACKGROUND OF THE INVENTION
Essentially all living organisms synthesize saturated fatty acids by the same biochemical mechanism. However, whereas vertebrates and yeast synthesize saturated fatty acids using either one or two multifunctional enzymes (i.e., type I fatty acid synthases, FASs), with the acyl carrier protein (ACP) being an integral part of the complex, most bacteria and plants synthesize saturated fatty acids through the use of a set of distinct enzymes that are each encoded by an individual gene (i.e., type II FASs). In the type II FAS system, ACP is also a distinct protein.
The initial step in the biosynthetic cycle of saturated fatty acids is performed by the enzyme FabH [Tsay et al.,
J. Biol. Chem
. 267:6807-68014 (1992), and U.S. Pat. No: 5,759,832, Issued Jun. 2, 1998, both of which are hereby incorporated by reference in their entireties] which catalyzes the condensation of malonyl-ACP with acetyl-COA. Malonyl-ACP is condensed with the growing-chain acyl-ACP in subsequent rounds by FabB synthase I or by FabF, synthase II. The next step is a ketoester reduction that is catalyzed by an NADPH-dependent &bgr;-ketoacyl-ACP reductase (FabG). A &bgr;-hydroxyacyl-ACP dehydrase (FabA, dehydrase I or FabZ, dehydrase II) catalyzes the subsequent dehydration forming trans-2-enoyl-ACP. FabI, an NADH-dependent enoyl-ACP reductase, then catalyzes the conversion of trans-2-enoyl-ACP to acyl-ACP to complete the elongation cycle. The addition of two carbon atoms per elongation cycle continues until palmitoyl-ACP is synthesized. Palmitoyl-ACP is one end-product of the pathway and acts as a feedback inhibitor for both FabH and FabI [Heath, et al,
J.Biol. Chem
. 271:1833-1836 (1996)].
Since an enoyl-ACP reductase catalyzes the final step in the biosynthetic pathway of saturated fatty acids, it is not surprising that it is also a key regulatory target for the pathway [Heath, and Rock,
J.Biol.Chem
. 271:1833-1836 (1996); Heath and Rock,
J.Biol.Chem
. 271:10996-11000 [(1996)]. Thus, pharmaceutical companies have placed considerable effort toward developing drugs that inhibit enoyl-ACP reductases and/or the reactions they catalyze. For example, the enoyl-ACP reductase of
Mycobacterium tuberculosis
(InhA) is a target for the drug isonaizid [Banerjee et al.,
Science
, 263:227 (1994)] whereas, both diazaborines [Baldock et al.,
Biochem. Phartmacol
., 55:1541 (1998)] and triclosan [McMurray et al.,
Nature
(London), 394:531 (1998) and Heath et al.,
J. Biol. Chem
., 273:30316 (1998)] inhibit the
Escherichia coli
enoyl-ACP reductase, FabI. All three drugs act through the formation of a high-affinity enzyme-NAD
+
-drug ternary complex [Heath et al.,
J. Biol. Chem
., 274:11110-11114 (1999) and Rozwarski et al.,
Science
, 279:98 (1998); Baldock et al.,
Science
, 274:2107 (1996); Levy et al.,
Nature
(London) 398:383 (1999); Stewart et al,
J. Mol. Biol
., 290:859 (1999); and Ward et al.,
Biochemistry
, 38:12514 (1999)]. Consistently, missense mutations resulting in single arnino acid substitutions in the active sites of the enoyl-ACP reductases prevent the formation of the ternary complexes and confer a resistant phenotype to bacteria expressing the mutant proteins [Banerjee et al.,
Science
, 263:227 (1994); McMurray et al.,
Nature
(London), 394:531 (1998); Heath et al.,
J. Biol. Chem
., 273:30316 (1998); Heath et al.,
J. Biol. Chem
., 274:11110-11114 (1999); and Bergler et al.,
J. Gen. Microbiol
., 138:2093 (1992) and Rouse et al.,
Antimicrobiol. Agents. Chem
., 39:2472 (1995)].
Unfortunately, the toxicity of boron severely limits the pharmaceutical application of diazaborines [Baldock et al,
Biochem. Phannacol
., 55:1541(1998)]. Triclosan, on the other hand, is widely employed as an antibacterial in consumer products for external use. Triclosan is a diphenyl ether (bis-phenyl) derivative, known as either 2,4,4′-Trichloro-2′-hydroxydiphenyl ether or 5-Chloro-2-(2,4-dichlorophenoxy) phenol, and is used as an antibacterial in antimicrobial creams, antiperspirants, body washes, cosmetics, deodorants, deodorant soaps, detergents, dish washing liquids, hand soaps, lotions, and toothpaste, as well as in plastics, polymers and textiles [see, Bhargava and Leonard,
Am. J. Infect. Control
, 24:209 (1996)]. However, the hydrophobic nature and chlorine content of triclosan makes it undesirable for internal use.
Bacterial infections remain among the most common and deadly causes of human disease. For example, Streptococci are known to cause otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis. In addition, virulent strains of
E. coli
can cause severe diarrhea, a condition which worldwide kills a million more people (3 million) every year than malaria [D. Leff,
BIOWORLD TODAY
, 9:1,3 (1998)]. Indeed, infectious diseases are the third leading cause of death in the United States and the leading cause of death worldwide [Binder et al.,
Science
284:1311-1313 (1999)].
Although, there was initial optimism in the middle of the 20th century that diseases caused by bacteria would be quickly eradicated, it has become evident that the so-called “miracle drugs” are not sufficient to accomplish this task. Indeed, antibiotic resistant pathogenic strains of bacteria have become common-place, and bacterial resistance to the new variations of these drugs appears to be outpacing the ability of scientists to develop effective chemical analogs of the existing drugs [See, Stuart B. Levy,
The Challenge of Antibiotic Resistance
, in
Scientific American
, 46-53 (March, 1998)]. Therefore, new approaches to drug development are necessary to combat the ever-increasing number of antibiotic-resistant pathogens.
Classical penicillin-type antibiotics effect a single class of proteins known as autolysins. Therefore, the development of new drugs which effect an alternative bacterial target protein would be desirable. Such a target protein ideally would be indispensable for bacterial survival. Thus the identification of a new bacterial enzyme that is required for fatty acid synthesis would be a prime candidate for such drug development.
Therefore, there is a need to identify new proteins that have enzymatic activities that are crucial for bacterial growth. There is also a need to provide immunogenic compositions containing such enzymes or fragments thereof. In addition, there is a need to develop methods for identifying drugs that interfere with such enzymes. Finally, there is a need to employ such procedures to develop new anti-bacterial drugs.
The citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.
SUMMARY OF THE INVENTION
The present invention provides two families of enzymes that can act as enoyl reductases. One such family shares a common amino acid consensus sequence, SEQ ID NO:45 and binds a flavin cofactor. This family of enoyl reductases is exemplified by the
Streptococcus pneumoniae
, FabK having an amino acid sequence of SEQ ID NO:2 and is naturally encoded by SEQ ID NO:1, as disclosed herein. The other family of enoyl reductases shares a common amino acid consensus sequence, SEQ ID NO:57 and like the previously disclosed FabI does not contain a flavin cofactor. This second family of enoyl reductases is exemplified by the
Campylobacter jejuni
FabL having an amino acid sequence of SEQ ID NO:52 and is naturally encoded by SEQ ID NO:51, as disclosed herein.
As discl

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