FabI

Details FabI FabI

1999-04-15

2004-06-22

Navarro, Mark (Department: 1645)

Chemistry: molecular biology and microbiology

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

C530S300000, C530S324000, C530S350000

Reexamination Certificate

active

06753172

ABSTRACT:

This invention relates, in part, to newly identified polynucleotides and polypeptides; variants and derivatives of these polynucleotides and polypeptides; processes for making these polynucleotides and these polypeptides, and their variants and derivatives; agonists and antagonists of the polypeptides; and uses of these polynucleotides, polypeptides, variants, derivatives, agonists and antagonists. In particular, in these and in other regards, the invention relates to polynucleotides and polypeptides of staphylococcal FabI enoyl-ACP reductase hereinafter referred to as “FABI”.
BACKGROUND OF THE INVENTION
Although the overall pathway of saturated fatty acid biosynthesis is similar in all organisms, the fatty acid synthase (FAS) systems vary considerably with respect to their structural organization. Thus in Type I FAS systems, found in vertebrates and yeasts, the necessary enzymes required for fatty acid synthesis are present on one or two polypeptide chains respectively. In contrast, in Type II systems found in most bacteria and plants, each step in the pathway is catalysed by a separate mono-functional enzyme. It would therefore appear that significant selectivity of inhibition of the bacterial and mammalian enzymes is possible.
FabI (previously designated EnvM) functions as an enoyl-acyl carrier protein (ACP) reductase (Bergler, et al, (1994),
J. Biol. Chem
. 269, 5493-5496) in the final step of the four reactions involved in each cycle of bacterial fatty acid biosynthesis.
The first step is catalysed by &bgr;-ketoacyl-ACP synthase, which condenses malonyl-ACP with acetyl-CoA (FabH, synthase III). In subsequent rounds malonyl-ACP is condensed with the growing-chain acyl-ACP (FabB and FabF, synthases I and II respectively).
The second step in the elongation cycle is ketoester reduction by NADPH-dependent &bgr;-ketoacyl-ACP reductase (FabG). Subsequent dehydration by &bgr;-hydroxyacyl-ACP dehydrase (either FabA or FabZ) leads to trans-2-enoyl-ACP which is in turn converted to acyl-ACP by NADH-dependent enoyl-ACP reductase (FabI). Further rounds of this cycle, adding two carbon atoms per cycle, eventually lead to palmitoyl-ACP (16C) where upon the cycle is stopped largely due to feedback inhibition of FabI by palmitoyl-ACP (Heath, et al, (1996),
J. Biol. Chem
. 271, 1833-1836). FabI is therefore a major biosynthetic enzyme which is also a key regulatory point in the overall synthetic pathway.
Early data suggested that there were two enoyl-ACP reductases in
E. coli
, one NADPH dependent and the other NADH dependent. However, more recent work has found no evidence for the NADPH dependent enzyme and FabI is the only enoyl ACP reductase identified in
E. coli
. (Heath, et al, (1995),
J. Biol. Chem
. 270, 26538-26542; Bergler, et al, (1994),
J. Biol. Chem
. 269, 5493-5496).
It has been shown that diazaborine antibiotics inhibit fatty acid, phospholipid and lipopolysaccharide (LPS) biosynthesis and it has also been shown that the antibacterial target of these compounds is FabI. For example derivative 2b18 from Grassberger, et al (1984)
J. Med Chem
27 947-953 has been shown to be a non-competitive inhibitor of FabI having a Ki=0.2 mM (Bergler, et al, (1994),
J. Biol. Chem
. 269, 5493-5496). The antibacterial activity of diazaborine derivatives against Gram-negatives and Gram positive organisms is well documented (Grassberger et al., J Med Chem. 1984 27, 947-953; Gronowitz et al., Acta Pharm Suecica, 1971 8 377; Wersch et al U.S. Pat. No. 2,533,918; Lam et al., J. Antimirob. Chemother. 1987 20 37-45).
Conditionally lethal FabI mutants have been constructed in
E. coli
and the FabI gene from
Salmonella typhimurium
complements this mutation. In addition, plasmids containing the FabI gene from diazaborine resistant
S. typhimurium
conferred diazaborine resistance in
E. coli
(Turnowsky, et al, (1989),
J. Bacteriol
., 171, 6555-6565) confirming FabI as the antibacterial target of diazaborines.
Inhibition of FabI either by diazaborine or by raising the temperature in an FabI temperature sensitive mutant to non-permissive conditions is lethal, thus demonstrating that FabI is essential to the survival of the organism (Bergler, et al, (1994),
J. Biol. Chem
. 269, 5493-5496). Laboratory generated point mutations in the FabI gene lead to diazaborine resistant
E. coli.
FabI is conserved in Gram negative organisms with 98% identity between
E. coli
and
S. typhimurium
FabI (Bergler, et al, (1992),
J. Gen. Microbiol
., 138, 2093-2100) and 75% identity between these proteins and
H. influenzae
FabI.
Staphylococcus aureus
FABI of the invention shows 54% similarity to the mycobacterial protein, InhA, which is highly conserved throughout mycobacteria including
M. tuberculosis. E. coli
FabI was found to be 34% identical, 57% similar to
Brassica napus
(rape seed) enoyl-ACP reductase and
S. aureus
FABI of the present invention was also 34% identical, 57% similar. Moreover, FABI of the present invention was found to be 44% identical, 64% similar over 252 amino acids to
E. coli
FabI. FABI of the present invention is only 27% identical, 48% similar to a mammalian 2,4-dienoyl-coenzyme A reductase. This mammalian homolog differs from FABI in that it is involved in the &bgr;-oxidation of polyunsaturated enoyl-CoAs and utilizes NADPH as cofactor rather than NADH. Therefore, there is significant potential for selective inhibition of FABI. Since there are no marketed antibiotics targeted against fatty acid biosynthesis it is likely that inhibitors of FABI will not be susceptible to current antibiotic resistance mechanisms. Moreover, this is a potentially broad spectrum target.
There is an unmet need for developing new classes of antibiotic compounds. Clearly, there is also a need for factors, such as FABI that may be used to screen compounds for antibiotic activity, such as a simple high through-put assay for screening inhibitors of FAS. Such factors may also be used to determine their roles in pathogenesis of infection, dysfunction and disease. Identification and characterization of such factors, which can play a role in preventing, ameliorating or correcting infections, dysfunctions or diseases are critical steps in making important discoveries to improve human health.
SUMMARY OF THE INVENTION
Toward these ends, and others, it is an object of the present invention to provide polypeptides, inter alia, that have been identified as novel FABI by homology between the amino acid sequence set out in
FIG. 1
[SEQ ID NO:2] and known amino acid sequences of other proteins such as
E. coli
FabI enoyl-ACP reductase and those afforementioned.
It is a further object of the invention, moreover, to provide polynucleotides that encode FABI, particularly polynucleotides that encode the polypeptide herein designated FABI.
In a particularly preferred embodiment of this aspect of the invention the polynucleotide comprises the region encoding FABI in the sequence set out in
FIG. 1
[SEQ ID NO:2].
In another particularly preferred embodiment of the present invention there is a novel FABI protein from
S. aureus
WCUH 29 comprising the amino acid sequence of SEQ ID NO:2, or a fragment, analogue or derivative thereof.
In accordance with this aspect of the invention there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressible by the
Staphylococcus aureus
WCUH 29 strain contained in NCIMB Deposit No. 40771.
In accordance with this aspect of the invention there are provided isolated nucleic acid molecules encoding FABI, particularly staphylococcal FABI, including mRNAs, cDNAs, genomic DNAs and, in further embodiments of this aspect of the invention, biologically. diagnostically, clinically or therapeutically useful variants, analogs or derivatives thereof, or fragments thereof, including fragments of the variants, analogs and derivatives.
Among the particularly preferred embodiments of this aspect of the invention are naturally occurring allelic variants of FABI.
In accordance with this aspect of the invention there are provided novel polypept

Affiliated with

Also associated with

Rating

FabI does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with FabI, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and FabI will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3355169