Crystal structure of rat GTP cyclohydrolase I feedback regulatory protein, GFRP11Edited by I. Wilson

Bader, Gerd; Schiffmann, Susanne; Herrmann, Anja; Fischer, Markus; Gütlich, Markus; Auerbach, Günter; Ploom, Tarmo; Bacher, Adelbert; Huber, Robert; Lemm, Thorsten

doi:10.1006/jmbi.2001.5011

Cited by 17 publications

(9 citation statements)

References 30 publications

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“…Recently the crystal structure of the free form of GFRP, which forms a pentameric ring similar to the GFRP pentamer in our stimulatory complex, has been reported (36). The rms deviation of C ␣ atoms (residues 2-83) between the free and GTPCHIbound forms of GFRP is small: 0.58 Å for monomer and 0.69 Å for pentamer.…”

Section: Resultsmentioning

confidence: 82%

Crystal structure of the stimulatory complex of GTP cyclohydrolase I and its feedback regulatory protein GFRP

Maita

Okada

Hatakeyama

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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In the presence of phenylalanine, GTP cyclohydrolase I feedback regulatory protein (GFRP) forms a stimulatory 360-kDa complex with GTP cyclohydrolase I (GTPCHI), which is the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin. The crystal structure of the stimulatory complex reveals that the GTPCHI decamer is sandwiched by two GFRP homopentamers. Each GFRP pentamer forms a symmetrical five-membered ring similar to ␤-propeller. Five phenylalanine molecules are buried inside each interface between GFRP and GTPCHI, thus enhancing the binding of these proteins. The complex structure suggests that phenylalanine-induced GTPCHI⅐GFRP complex formation enhances GTPCHI activity by locking the enzyme in the active state.

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Section: Resultsmentioning

confidence: 82%

Crystal structure of the stimulatory complex of GTP cyclohydrolase I and its feedback regulatory protein GFRP

Maita

Okada

Hatakeyama

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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“…In mammals, feedback inhibition of GCHI by the end product tetrahydrobiopterin is mediated by a pentameric GCHI feedback-regulatory protein (GFRP) that binds specifically to each of the two faces of the toroid-shaped GCHI decamer (35,36). That plant GCHI has primary and quaternary structures so unlike those of mammals makes it improbable that the plant enzyme has the same type of feedback-inhibitory mechanism.…”

Section: Discussionmentioning

confidence: 99%

Folate synthesis in plants: The first step of the pterin branch is mediated by a unique bimodular GTP cyclohydrolase I

Basset

Quinlivan

Ziemak

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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GTP cyclohydrolase I (GCHI) mediates the first and committing step of the pterin branch of the folate-synthesis pathway. In microorganisms and mammals, GCHI is a homodecamer of Ϸ26-kDa subunits. Genomic approaches identified tomato and Arabidopsis cDNAs specifying Ϸ50-kDa proteins containing two GCHI-like domains in tandem and indicated that such bimodular proteins occur in other plants. Neither domain of these proteins has a full set of the residues involved in substrate binding and catalysis in other GCHIs. The tomato and Arabidopsis cDNAs nevertheless encode functional enzymes, as shown by complementation of a yeast fol2 mutant and by assaying GCHI activity in extracts of complemented yeast cells. Neither domain expressed separately had GCHI activity. Recombinant tomato GCHI formed dihydroneopterin triphosphate as reaction product, as do other GCHIs, but unlike these enzymes it did not show cooperative behavior and was inhibited by its substrate. Denaturing gel electrophoresis verified that the bimodular GCHI polypeptide is not cleaved in vivo into its component domains, and size-exclusion chromatography indicated that the active enzyme is a dimer. The deduced tomato and Arabidopsis GCHI polypeptides lack overt targeting sequences and thus are presumably cytosolic, in contrast to other plant folate-synthesis enzymes, which are mitochondrial proteins with typical signal peptides. GCHI mRNA and protein are strongly in expressed unripe tomato fruits, implying that fruit folate is made in situ rather than imported. As ripening advances, GCHI expression declines sharply, and folate content drops, suggesting that folate synthesis fails to keep pace with turnover.T etrahydrofolate and its derivatives (folates) are essential cofactors for one-carbon transfer reactions in all organisms. Similar to bacteria and yeasts, plants make folates de novo from pterin, p-aminobenzoate (PABA), and glutamate moieties (1, 2). In contrast, humans and other mammals lack a complete folatesynthesis pathway and thus need dietary folate. Because plant foods are major folate sources, and folate deficiency is a global health problem, enhancing plant folate content is a prime target for metabolic engineering (1, 3). This engineering demands knowledge of the biosynthetic pathway.The plant folate-synthesis pathway is not understood fully, but is most probably similar to that in bacteria (1, 2). The pterin hydroxymethyldihydropteroate is formed from GTP, and PABA from chorismate. The pterin and PABA units are condensed, glutamylated, and reduced to give tetrahydrofolate, and a polyglutamyl tail is added (4). Plant genes and enzymes for the final five steps have been characterized (5, 6), and the enzymes have been shown to be mitochondrial (7,8). Far less is known for plants about the early steps that produce the pterin and PABA moieties.The first step of pterin synthesis is of special interest, because it commits GTP to pterin production and is considered to control flux into the pathway (9, 10). This step, mediated by GTP cyclohydrolase I (GC...

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“…To date, only the rat GFRP crystal structure has been solved, but sequence comparison shows 95% amino acid sequence homology with the human protein (Bader et al, 2001) (Maita et al, 2002) (Maita et al, 2004) ( Multi-species comparison of GFRP amino acid sequences. The boxes frame the amino acid residues that make contact with GTPCH1 at the GTPCH1-GFRP interface.…”

Section: Gtpch1 Feedback Regulatory Protein: Gfrpmentioning

confidence: 99%

“…Zinc is essential for GTPCH1 activity (Auerbach et al, 2001). Studies suggested that divalent cations including magnesium, manganese, calcium and zinc have the capacity to modulate GTPCH1 activity (Hatakeyama et al, 1989) (Steinmetz et al, 1998) (Suzuki et al, 2004).…”

Section: (2) Cofactors: Divalent Cationsmentioning

confidence: 99%

Investigating the interaction between GTP-cyclohydrolase1 and its feedback regulatory protein

et al. 2012

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GTP-cyclohydrolase-1 (GTPCH1) catalyses the rate-limiting step in the biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor for enzymes including aromatic amino acid hydroxylases and nitric oxide synthases. Strategies that increase vascular BH4 biosynthesis represent a promising therapeutic approach for the treatment of endothelial dysfunction. GTPCH1 is subject to feedback and feed-forward regulation by BH4 and L-phenylalanine (L-phe) respectively, via an allosteric protein interaction with GTPCH1 feedback regulatory protein (GFRP). The aim of this thesis was to investigate the GTPCH1-GFRP interaction using recombinant proteins, and to validate the functional significance of the interaction in the vasculature using animal models. Human GTPCH1 and GFRP proteins were recombinantly expressed. Studies compared the activity and protein interactions of native GTPCH1 with a truncated mutant. A kinetic GTPCH1 activity assay was modified to enable a high-throughput screen of a fragment library. GTPCH1-GFRP interactions were assessed using surface plasmon resonance (SPR). Finally, in-vivo and ex-vivo functional studies in rodents investigated the effects of L-phe on vascular function and BH4 levels. Studies using recombinant proteins revealed the activity of truncated GTPCH1 exceeds that of native GTPCH1. A high-throughput screen successfully identified four compounds that modulate GTPCH1 activity. Biophysical analysis (SPR) demonstrated that both native and truncated GTPCH1 bind to GFRP in the absence of natural ligands. The kinetics and binding rate constants have been reported for the first time. In functional studies, oral L-phe supplementation in rodents led to a rise of BH4 levels within aortic tissue, and reversed vascular dysfunction observed in vessels obtained from spontaneously hypertensive rats. This thesis demonstrates that modulation of the GTPCH1-GFRP interactions represents a novel therapeutic target to regulate endogenous BH4 levels. SPR data suggests that GTPCH1 and 2 GFRP are constitutively bound in-vivo and indicate that the N-terminal region of GTPCH1 may directly interact with GFRP and modulate basal enzyme activity. The functional effects of L-phe on vascular BH4 levels and function, validate the potential of the GTPCH1-GFRP pathway as a therapeutic target for cardiovascular disease. 3

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Crystal structure of rat GTP cyclohydrolase I feedback regulatory protein, GFRP11Edited by I. Wilson

Cited by 17 publications

References 30 publications

Crystal structure of the stimulatory complex of GTP cyclohydrolase I and its feedback regulatory protein GFRP

Crystal structure of the stimulatory complex of GTP cyclohydrolase I and its feedback regulatory protein GFRP

Folate synthesis in plants: The first step of the pterin branch is mediated by a unique bimodular GTP cyclohydrolase I

Investigating the interaction between GTP-cyclohydrolase1 and its feedback regulatory protein

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