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Crystal structure of the yeast His6 enzyme suggests a reaction mechanism.

ABSTRACT: The Saccharomyces cerevisiae His6 gene codes for the enzyme phosphoribosyl-5-amino-1-phosphoribosyl-4-imidazolecarboxamide isomerase, catalyzing the fourth step in histidine biosynthesis. To get an insight into the structure and function of this enzyme, we determined its X-ray structure at a resolution of 1.30 A using the anomalous diffraction signal of the protein's sulphur atoms at 1.77 A wavelength. His6 folds in an (alpha/beta)8 barrel similar to HisA, which performs the same function in bacteria and archaea. We found a citrate molecule from the buffer bound in a pocket near the expected position of the active site and used it to model the open form of the substrate (phosphoribulosyl moiety), which is a reaction intermediate. This model enables us to identify catalytic residues and to propose a reaction mechanism where two aspartates act as acid/base catalysts: Asp134 as a proton donor for ring opening, and Asp9 as a proton acceptor and donor during enolization of the aminoaldose. Asp9 is conserved in yeast His6 and bacterial or archaeal HisA sequences, and Asp134 has equivalents in both HisA and TrpF, but they occur at a different position in the protein sequence.

SUBMITTER: Quevillon-Cheruel S

PROVIDER: S-EPMC2242542 | biostudies-literature | 2006 Jun

REPOSITORIES: biostudies-literature

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Crystal structure of the yeast His6 enzyme suggests a reaction mechanism.

Quevillon-Cheruel Sophie S Leulliot Nicolas N Graille Marc M Blondeau Karine K Janin Joel J van Tilbeurgh Herman H

Protein science : a publication of the Protein Society 20060601 6

The Saccharomyces cerevisiae His6 gene codes for the enzyme phosphoribosyl-5-amino-1-phosphoribosyl-4-imidazolecarboxamide isomerase, catalyzing the fourth step in histidine biosynthesis. To get an insight into the structure and function of this enzyme, we determined its X-ray structure at a resolution of 1.30 A using the anomalous diffraction signal of the protein's sulphur atoms at 1.77 A wavelength. His6 folds in an (alpha/beta)8 barrel similar to HisA, which performs the same function in bac ...[more]

PMID: 16731983

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Project description:AMPylation is a prevalent posttranslational modification that involves the addition of adenosine monophosphate (AMP) to proteins. Exactly how Huntingtin-associated yeast-interacting protein E (HYPE), as the first human protein, is involved in the transformation of the AMP moiety to its substrate target protein (the endoplasmic reticulum chaperone binding to immunoglobulin protein (BiP)) is still an open question. Additionally, a conserved glutamine plays a vital key role in the AMPylation reaction in most filamentation processes induced by the cAMP (Fic) protein. In the present work, the detailed catalytic AMPylation mechanisms in HYPE were determined based on the density functional theory (DFT) method. Molecular dynamics (MD) simulations were further used to investigate the exact role of the inhibitory glutamate. The metal center, Mg2+, in HYPE has been examined in various coordination configurations, including 4-coordrinated, 5-coordinated and 6-coordinated. DFT calculations revealed that the transformation of the AMP moiety of HYPE with BiP followed a sequential pathway. The model with a 4-coordinated metal center had a barrier of 14.7 kcal/mol, which was consistent with the experimental value and lower than the 38.7 kcal/mol barrier of the model with a 6-coordinated metal center and the 31.1 kcal/mol barrier of the model with a 5-coordinated metal center. Furthermore, DFT results indicated that Thr518 residue oxygen directly attacks the phosphorus, while the His363 residue acts as H-bond acceptor. At the same time, an MD study indicated that Glu234 played an inhibitory role in the α-inhibition helix by regulating the hydrogen bond interaction between Arg374 and the Pγ of the ATP molecule. The revealed sequential pathway and the inhibitory role of Glu234 in HYPE were inspirational for understanding the catalytic and inhibitory mechanisms of Fic-mediated AMP transfer, paving the way for further studies on the physiological role of Fic enzymes.

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Crystal structure of the yeast His6 enzyme suggests a reaction mechanism.

Publications

Crystal structure of the yeast His6 enzyme suggests a reaction mechanism.

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