Riboflavin synthase genes and enzymes and methods of use

Details Riboflavin synthase genes and enzymes and methods of use Riboflavin synthase genes and enzymes and methods of use

1999-03-26

2002-02-26

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

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

C435S410000, C435S006120, C536S023200

Reexamination Certificate

active

06350597

ABSTRACT:

FIELD OF THE INVENTION
This invention is in the field of plant and fungal molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding proteins involved in the riboflavin biosynthetic pathway of plants or fungi.
BACKGROUND OF THE INVENTION
Riboflavin, vitamin B
2
, is the precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), essential cofactors for a number of mainstream metabolic enzymes that mediate hydride, oxygen, and electron transfer reactions. Riboflavin-dependent enzymes include succinate dehydrogenase, NADH dehydrogenase, ferredoxin-NADP
+
oxidoreductase, acyl-CoA dehydrogenase, and the pyruvate dehydrogenase complex. Consequently, fatty acid oxidation, the TCA cycle, mitochondrial electron-transport, photosynthesis, and numerous other cellular processes are critically dependent on either FMN or FAD as prosthetic groups. Other notable flavoproteins include glutathione reductase, glycolate oxidase, P450 oxido-reductase, squalene epoxidase, dihydroorotate dehydrogenase, and &agr;-glycerophosphate dehydrogenase. Genetic disruption of riboflavin biosynthesis in
E. coli
(Richter et al.,
J. Bacteriol
. 174:4050-4056 (1992)) and
S. cerevisiae
(Santos et al.,
J. Biol. Chem
. 270:437-444 (1995)) results in a lethal phenotype that is only overcome by riboflavin supplementation. This is not surprising, considering the ensemble of deleterious pleiotropic effects that would occur with riboflavin deprivation.
Riboflavin is synthesized by plants and numerous microorganisms, including bacteria and fungi (Bacher, A.,
Chemistry and Biochemistry of Flavoproteins
(Müller, F., ed.) vol. 1, pp. 215-259, Chemical Rubber Co., Boca Raton, Fla. (1990)). Since birds, mammals, and other higher organisms are unable to synthesize the vitamin and, instead, rely on its dietary ingestion to meet their metabolic needs, the enzymes that are responsible for riboflavin biosynthesis are potential targets for future antibiotics, fungicides, and herbicides. Moreover, it is possible that the distantly-related plant and microbial enzymes have distinct characteristics that could be exploited in the development of potent organism-specific inhibitors. Thus, a detailed understanding of the structure, mechanism, kinetics, and substrate-binding properties of the riboflavin biosynthetic enzyme(s), from plants for example, would serve as a starting point for the rational design of chemical compounds that might be useful as herbicides. Having the authentic plant protein(s) in hand would also provide a valuable tool for the in vitro screening of chemical libraries in search of riboflavin biosynthesis inhibitors.
Bacterial and fungal riboflavin biosynthesis has been intensively studied for more than four decades (For recent reviews, see Bacher, A.,
Chemistry and Biochemistry of Flavoproteins
(Müller, F., ed.) vol. I, pp. 215-259 and 293-316 Chemical Rubber Co., Boca Raton, Fla. (1990)). The synthetic pathway consists of seven distinct enzyme catalyzed reactions, with guanosine 5′-triphosphate (GTP) and ribulose 5-phosphate the ultimate precursors. While the second and third steps of riboflavin biosynthesis occur in opposite order in bacteria and fungi, the remaining pathway intermediates are identical in both microorganisms. Structurally and mechanistically, the last two reactions in the pathway, namely, those catalyzed by 6,7-dimethyl-8-ribityllumazine synthase (LS) and riboflavin synthase (RS), are best characterized. In
B. subtilis
, these two enzymes are physically associated with each other in a huge spherical particle with a combined molecular mass of about 1 MDa (Bacher et al.,
J. Biol Chem
. 255:632-637 (1980); Ritsert et al.,
J. Mol. Biol
. 253, 151-167 (1995); Bacher et al.,
Biochem. Soc. Trans
. 24(1):89-94 (1996)); the X-ray structure of the bifunctional protein complex has been determined at 3.3 angstrom resolution (Ladenstein et al.,
J. Mol. Biol
203:1045-1070). The LS/RS complex consists of 60 LS subunits that are organized into 12 pentamers to form a hollow icosahedral capsid. Encaged in the central core of this structure resides a single molecule of RS, a trimer of three identical subunits. Kinetic studies reveal that the compartmentation of the two enzymes within the complex improves the overall catalytic efficiency of riboflavin production at low substrate concentrations, presumably via “substrate channeling” (Kis et al.,
J. Biol. Chem
. 270:16788-16795 (1995)). Although a bifunctional LS/RS complex has not been observed in other microorganisms, it was recently shown that the native
E. coli
LS also exists in vivo as a hollow icosahedral capsid of 60 identical subunits (Mörtl et al.,
J. Biol. Chem
. 271:33201-33207 (1996)).
LS, the penultimate enzyme of riboflavin biosynthesis, catalyzes the condensation of 3,4-dihydroxy-2-butanone 4-phosphate with 4-ribitylamino-5-amino-2,6-dihydroxypyrimidine (RAADP) to yield 1 mol each of orthophosphate and 6,7-dimethyl-8-(1′-D-ribityl)-lumazine (DMRL). The latter is the immediate precursor of riboflavin. LS-encoding genes have been cloned from numerous microorganisms, including
E. coli
(Taura et al.,
Mol. Gen. Genet
. 234:429-432 (1992)),
A. pleuropneumoniae
(Fuller et al.,
J. Bacteriol
. 177:7265-7270 (1995)),
P. phosphoreum
(Lee et al.,
J. Bacteriol
. 176:2100-2104 (1994)),
B. subtilis
(Mironov et al.,
Dokl. Akad Nauk SSSR
305:482-487 (1989)), and
S. cerevisiae
(Garcia-Ramirez et al.,
J. Biol. Chem
. 270:23801-23807 (1995)). In all cases, the subunit molecular mass of the LS gene product is small, ranging in size from ~16-17 kDa.
While the various LS homologs all share certain structural features in common, their overall homology at the primary amino acid sequence level is rather poor. For example, as determined with the Genetics Computer Group Gap program (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.), the
E. coli
LS is only 58%, 65%, 53%, and 36% identical to the homologous proteins of
A. pleuropneumoniae, P. phosphoreum, B. subtilis
and
S. cerevisiae
, respectively. Indeed, pairwise comparisons of these five proteins reveal that the two most similar homologs share only 72% identity.
The terminal step of riboflavin biosynthesis is mediated by RS. This enzyme catalyzes the dismutation of two molecules of DMRL to yield 1 mol of riboflavin and RAADP. That the latter product is also one of the substrates of LS explains in part the enhanced catalytic efficiency of the
B. subtilis
LS/RS complex noted above. Although the crystal structure of RS remains to be determined, it is surmised that the native bacterial (Bacher et al.,
J. Biol. Chem
. 255:632-637 (1980)) and fungal (Santos et al.,
J. Biol. Chem
. 270:437-444 (1995)) proteins are trimers, each consisting of three identical ~25 kDa subunits. To date, RS has only been cloned from about a dozen microorganisms, and all of the species that have been examined exhibit marked internal homology in their N-terminal and C-terminal domains (Schott et al.,
J. Biol. Chem
. 265:4204-4209 (1990); Santos et al.,
J. Biol. Chem
. 270:437-444 (1995)). Based on these observations, it has been suggested that the two halves of the RS protomer have arisen through gene duplication, and that each contains a substrate-binding site for DMRL.
Despite this structural similarity, however, the overall sequence homology of the various RS proteins is extremely limited. Thus, the
E. coli
RS protein is only 32%, 36%, 35%, and 31% identical to its counterparts in
S. cerevisiae, P. phosphoreum., B. subtilis
, and
P. leiognathi
; the GenBank accession numbers for the latter four proteins are Z21621, L11391, X51510 and M90094, respectively.
With the exception of GTP cyclohydrolase II, the first committed enzyme of riboflavin biosynthesis, virtually nothing is known about the riboflavin biosynthetic machinery of higher plants. The gene for this protein was recently cloned from an arabidopsis cDNA library (Kobayashi et al.,
Gene
160:303-304 (1995)). The protein sequence of the cloned plant g

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