Purification and characterization of 6‐pyruvoyl tetrahydropterin synthase from salmon liver

Hasler, Thomas; Curtius, Hans−Christoph

doi:10.1111/j.1432-1033.1989.tb14635.x

Cited by 17 publications

(9 citation statements)

References 23 publications

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“…A clear difference between PTPS and SSCR activities by E. coli enzyme is that the former required Mg 2+ and reducing agents (data not shown), while the latter did not. The role of Mg 2+ and reducing agents was also well established in higher animal enzymes [18,23–25].…”

Section: Discussionmentioning

confidence: 96%

Escherichia coli 6‐pyruvoyltetrahydropterin synthase ortholog encoded by ygcM has a new catalytic activity for conversion of sepiapterin to 7,8‐dihydropterin

Woo¹,

Hwang²,

Kim³

et al. 2002

FEBS Letters

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The putative gene (ygcM) of Escherichia coli was veri¢ed in vitro to encode the ortholog of 6-pyruvoyltetrahydropterin synthase (PTPS). Unexpectedly, the enzyme was found to convert sepiapterin to 7,8-dihydropterin without any cofactors. The enzymatic product 7,8-dihydropterin was identi¢ed by HPLC and mass spectrometry analyses, suggesting a novel activity of the enzyme to cleave the C6 side chain of sepiapterin. The optimal activity occurred at pH 6.5^7.0. The reaction rate increased up to 3.2-fold at 60^80 ‡C, re£ecting the thermal stability of the enzyme. The reaction required no metal ion and was activated slightly by low concentrations (1^5 mM) of EDTA. The apparent K m value for sepiapterin was determined as 0.92 mM and the V max value was 151.3 nmol/min/ mg. The new catalytic function of E. coli PTPS does not imply any physiological role, because sepiapterin is not an endogenous substrate of the organism. The same activity, however, was also detected in a PTPS ortholog of Synechocystis sp. PCC 6803 but not signi¢cant in Drosophila and human enzymes, suggesting that the activity may be prevalent in bacterial PTPS orthologs. ß

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Section: Discussionmentioning

confidence: 96%

Escherichia coli 6‐pyruvoyltetrahydropterin synthase ortholog encoded by ygcM has a new catalytic activity for conversion of sepiapterin to 7,8‐dihydropterin

Woo¹,

Hwang²,

Kim³

et al. 2002

FEBS Letters

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“…The critical prop erty for the purification was the heat insta bility of the human pituitary gland enzyme, in contrast to the human [15], rat [18] and salmon liver [16], and Drosophila head [17] enzymes. Another important difference was the chromatography on hydroxylapatite fol lowing ammonium sulfate precipitation, which resulted in a very high loss of activity.…”

Section: Resultsmentioning

confidence: 99%

“…Dro sophila melanogaster [9], silkworm [10], bo vine adrenal medulla [11], rat liver [12] and human brain and liver [13,14], The enzyme has been characterized from human [ 15] and salmon liver [ 16] as well as from D. melano gaster [17]. Recently, PTPS was purified Assay for PTPS Activity Enzyme activity was measured as described [15], except that the incubation time was reduced to 20 min.…”

Section: Ptps Is Widely Distributed In Mammalsmentioning

confidence: 99%

Purification and Characterization of 6-Pyruvoyl Tetrahydropterin Synthase from Human Pituitary Gland

Guzman

Redweik²,

Schoedon³

et al. 1992

Enzyme

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6-Pyruvoyl tetrahydropterin synthase, the enzyme that catalyses the conversion of 7,8-dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin, was purified 3,330- fold from human pituitary gland with an overall recovery of 30%. The native enzyme has a molecular mass of 68 kD and consists of four identical subunits of 16.5 kD. The pH optimum of the enzyme in Tris/HCl buffer is 7.5. The enzyme is dependent on Mg^2+ and NADPH and has a Michaelis-Menten constant of 10 μM for its natural substrate, 7,8-dihydroneopterin triphosphate. The isoelectric point of the human enzyme is 4.3-4.6. The human pituitary gland enzyme is heat instable in contrast to the enzymes from human, rat and salmon liver, and Drosophila head. The amino acid composition showed remarkably high content of acidic amino acids Asp and Glu. The N-terminus was found to be blocked.

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“…Suppression of the ' H 2 0 signals was achieved by homo-gated decoupling. The sample was prepared using a buffer system optimized for PPH, synthase [34], which was obtained by dissolving z 0.5 mg NH2TP in z O . 3 ml phosphate pH 7.6 containing 8 mM MgC12 and 1 mM dithioerythritol.…”

Section: Instrumentation For ' H -N M R and Sample Preparationmentioning

confidence: 99%

“…Since human PPH, synthase showed a much lower specific activity than the salmon enzyme [33], this enzyme was purified from salmon liver [34].…”

Section: Enzymesmentioning

confidence: 99%

¹H‐NMR and mass spectrometric studies of tetrahydropterins

Ghisla

Kuster

Steinerstauch

et al. 1990

European Journal of Biochemistry

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The conversion of dihydroneopterin triphosphate in the presence of 6-pyruvoyl tetrahydropterin synthase was followed by 'H-NMR spectroscopy. The interpretation of the spectra of the product is unequivocal: they show formation of a tetrahydropterin system carrying a stereospecifically oriented substituent at the asymmetric C(6) atom. The spectra are compatible with formation of a (3')-CH3 function, and with complete removal of the 1' and 2' hydrogens of dihydroneopterin triphosphate. The fast-atom-bombardment/mass spectrometry study of the same product yields a [M+H]+ ion at m/z 238 compatible with the structure of 6-pyruvoyl tetrahydropterin. The data support the proposed structure of 6-pyruvoyl tetrahydropterin as a key intermediate in the biosynthesis of tetrah ydrobiopterin.The biosynthetic pathway of tetrahydrobiopterin (BH,) has been elucidated in its prominent features in the past few years by the convergent efforts of several groups [I -91. The following sequence is now being widely accepted: GTP 4 7,X-dihydroneopterin triphosphate (NH2TP) + h-pyruvoyl-5,6,7,%tetrahydropterin (PPH,) 6-lactoyl-5,6,7,8-tetrahydropterin or 6-hydroxy-acetonoyl-5,6,7,X-tetrahydropterin + tetrahydrobiopterin (BH,).The enzymes involved have also been purified and characterized 14, 10-281. While the chemical structures and some of the properties of these intermediates are known, the mechanisms by which 7,s-dihydroneopterin triphosphate (NH2TP) and 6-pyruvoyl-5,6,7,8-tetrahydropterin (PPH4) are formed have not yet been elucidated. The first step, the conversion of GTP to NH2TP, is an apparently rather complicated process catalyzed by a single enzyme, GTP cyclohydrolase I, and involving ring opening of GTP, extrusion of a formyl equivalent, rearrangement of the ribosyl moiety and reclosure to form a six-membered ring [19, 201. The second step also involves two chemically distinct processes, the elimination of triphosphate from the terminal (C3') position of the C(6) side chain (23, 21 -231, and an internal, Amadori-type rearrangement during which the N(5)-C(6) double bond of NH2TP is reduced at the expense of oxidation of C(l')-OH to C(l')=O 13, 12,21,[23][24][25][26] A further major issue at the onset of our work was the chemical identity of PPH, and its properties. The intermediacy of this molecule in BH, biosynthesis had been proposed by several groups 16, 7, 23 -251, but its high instability and the difficulties in obtaining substantial quantities of material have hindered structural studies. In fact, the only feasible procedure for its preparation requires purified 6-pyruvoyl tetrahydropterin synthase and purified NH2TP [27], the latter also having to be prepared enzymatically from GTP [28]. Furthermore, the evidence presented for its structure was deduced from trapping experiments involving modifications of the pyruvoyl function such as reduction with NaBH4 or enzymatically with NADPH to BH4 [3, 4, 6, 8, 22, 23, 25, 26, 291. In the present work we report detailed 'H-NMR and fastatom-bombardment/mass spectrometry (FAB/MS) ...

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Purification and characterization of 6‐pyruvoyl tetrahydropterin synthase from salmon liver

Cited by 17 publications

References 23 publications

Escherichia coli 6‐pyruvoyltetrahydropterin synthase ortholog encoded by ygcM has a new catalytic activity for conversion of sepiapterin to 7,8‐dihydropterin

Escherichia coli 6‐pyruvoyltetrahydropterin synthase ortholog encoded by ygcM has a new catalytic activity for conversion of sepiapterin to 7,8‐dihydropterin

Purification and Characterization of 6-Pyruvoyl Tetrahydropterin Synthase from Human Pituitary Gland

¹H‐NMR and mass spectrometric studies of tetrahydropterins

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Purification and characterization of 6‐pyruvoyl tetrahydropterin synthase from salmon liver

Cited by 17 publications

References 23 publications

Escherichia coli 6‐pyruvoyltetrahydropterin synthase ortholog encoded by ygcM has a new catalytic activity for conversion of sepiapterin to 7,8‐dihydropterin

Escherichia coli 6‐pyruvoyltetrahydropterin synthase ortholog encoded by ygcM has a new catalytic activity for conversion of sepiapterin to 7,8‐dihydropterin

Purification and Characterization of 6-Pyruvoyl Tetrahydropterin Synthase from Human Pituitary Gland

1H‐NMR and mass spectrometric studies of tetrahydropterins

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¹H‐NMR and mass spectrometric studies of tetrahydropterins