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Crystal structure and mechanism of human L-arginine:glycine amidinotransferase: a mitochondrial enzyme involved in creatine biosynthesis.

ABSTRACT: L-arginine:glycine amidinotransferase (AT) catalyses the committed step in creatine biosynthesis by formation of guanidinoacetic acid, the immediate precursor of creatine. We have determined the crystal structure of the recombinant human enzyme by multiple isomorphous replacement at 1.9 A resolution. A telluromethionine derivative was used in sequence assignment. The structure of AT reveals a new fold with 5-fold pseudosymmetry of circularly arranged betabeta alphabeta-modules. These enclose the active site compartment, which is accessible only through a narrow channel. The overall structure resembles a basket with handles that are formed from insertions into the betabeta alphabeta-modules. Binding of L-ornithine, a product inhibitor, reveals a marked induced-fit mechanism, with a loop at the active site entrance changing its conformation accompanied by a shift of an alpha-helix by -4 A. Binding of the arginine educt to the inactive mutant C407A shows a similar mode of binding. A reaction mechanism with a catalytic triad Cys-His-Asp is proposed on the basis of substrate and product bound states.

SUBMITTER: Humm A

PROVIDER: S-EPMC1169963 | biostudies-other | 1997 Jun

REPOSITORIES: biostudies-other

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Crystal structure and mechanism of human L-arginine:glycine amidinotransferase: a mitochondrial enzyme involved in creatine biosynthesis.

Humm A A Fritsche E E Steinbacher S S Huber R R

The EMBO journal 19970601 12

L-arginine:glycine amidinotransferase (AT) catalyses the committed step in creatine biosynthesis by formation of guanidinoacetic acid, the immediate precursor of creatine. We have determined the crystal structure of the recombinant human enzyme by multiple isomorphous replacement at 1.9 A resolution. A telluromethionine derivative was used in sequence assignment. The structure of AT reveals a new fold with 5-fold pseudosymmetry of circularly arranged betabeta alphabeta-modules. These enclose the ...[more]

PMID: 9218780

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Project description:AimsCreatine buffers cellular adenosine triphosphate (ATP) via the creatine kinase reaction. Creatine levels are reduced in heart failure, but their contribution to pathophysiology is unclear. Arginine:glycine amidinotransferase (AGAT) in the kidney catalyses both the first step in creatine biosynthesis as well as homoarginine (HA) synthesis. AGAT-/- mice fed a creatine-free diet have a whole body creatine-deficiency. We hypothesized that AGAT-/- mice would develop cardiac dysfunction and rescue by dietary creatine would imply causality.Methods and resultsWithdrawal of dietary creatine in AGAT-/- mice provided an estimate of myocardial creatine efflux of ?2.7%/day; however, in vivo cardiac function was maintained despite low levels of myocardial creatine. Using AGAT-/- mice naïve to dietary creatine we confirmed absence of phosphocreatine in the heart, but crucially, ATP levels were unchanged. Potential compensatory adaptations were absent, AMPK was not activated and respiration in isolated mitochondria was normal. AGAT-/- mice had rescuable changes in body water and organ weights suggesting a role for creatine as a compatible osmolyte. Creatine-naïve AGAT-/- mice had haemodynamic impairment with low LV systolic pressure and reduced inotropy, lusitropy, and contractile reserve. Creatine supplementation only corrected systolic pressure despite normalization of myocardial creatine. AGAT-/- mice had low plasma HA and supplementation completely rescued all other haemodynamic parameters. Contractile dysfunction in AGAT-/- was confirmed in Langendorff perfused hearts and in creatine-replete isolated cardiomyocytes, indicating that HA is necessary for normal cardiac function.ConclusionsOur findings argue against low myocardial creatine per se as a major contributor to cardiac dysfunction. Conversely, we show that HA deficiency can impair cardiac function, which may explain why low HA is an independent risk factor for multiple cardiovascular diseases.

Dataset Information

Crystal structure and mechanism of human L-arginine:glycine amidinotransferase: a mitochondrial enzyme involved in creatine biosynthesis.

Publications

Crystal structure and mechanism of human L-arginine:glycine amidinotransferase: a mitochondrial enzyme involved in creatine biosynthesis.

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