Dataset Information

Structure and Mechanism of a Viral Collagen Prolyl Hydroxylase.

ABSTRACT: The Fe(II)- and 2-oxoglutarate (2-OG)-dependent dioxygenases comprise a large and diverse enzyme superfamily the members of which have multiple physiological roles. Despite this diversity, these enzymes share a common chemical mechanism and a core structural fold, a double-stranded ?-helix (DSBH), as well as conserved active site residues. The prolyl hydroxylases are members of this large superfamily. Prolyl hydroxylases are involved in collagen biosynthesis and oxygen sensing in mammalian cells. Structural-mechanistic studies with prolyl hydroxylases have broader implications for understanding mechanisms in the Fe(II)- and 2-OG-dependent dioxygenase superfamily. Here, we describe crystal structures of an N-terminally truncated viral collagen prolyl hydroxylase (vCPH). The crystal structure shows that vCPH contains the conserved DSBH motif and iron binding active site residues of 2-OG oxygenases. Molecular dynamics simulations are used to delineate structural changes in vCPH upon binding its substrate. Kinetic investigations are used to report on reaction cycle intermediates and compare them to the closest homologues of vCPH. The study highlights the utility of vCPH as a model enzyme for broader mechanistic analysis of Fe(II)- and 2-OG-dependent dioxygenases, including those of biomedical interest.

SUBMITTER: Longbotham JE

PROVIDER: S-EPMC4613865 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Structure and Mechanism of a Viral Collagen Prolyl Hydroxylase.

Longbotham James E JE Levy Colin C Johannissen Linus O LO Tarhonskaya Hanna H Jiang Shuo S Loenarz Christoph C Flashman Emily E Hay Sam S Schofield Christopher J CJ Scrutton Nigel S NS

Biochemistry 20150930 39

The Fe(II)- and 2-oxoglutarate (2-OG)-dependent dioxygenases comprise a large and diverse enzyme superfamily the members of which have multiple physiological roles. Despite this diversity, these enzymes share a common chemical mechanism and a core structural fold, a double-stranded β-helix (DSBH), as well as conserved active site residues. The prolyl hydroxylases are members of this large superfamily. Prolyl hydroxylases are involved in collagen biosynthesis and oxygen sensing in mammalian cells ...[more]

PMID: 26368022

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Project description:BackgroundWe describe a method for specific, quantitative and quick detection of human collagen prolyl 4-hydroxylase (C-P4H), the key enzyme for collagen prolyl-4 hydroxylation, in crude samples based on a sandwich ELISA principle. The method is relevant to active C-P4H level monitoring during recombinant C-P4H and collagen production in different expression systems. The assay proves to be specific for the active C-P4H alpha2beta2 tetramer due to the use of antibodies against its both subunits. Thus in keeping with the method C-P4H is captured by coupled to an anti-alpha subunit antibody magnetic beads and an anti-beta subunit antibody binds to the PDI/beta subunit of the protein. Then the following holoenzyme detection is accomplished by a goat anti-rabbit IgG labeled with alkaline phosphatase which AP catalyzes the reaction of a substrate transformation with fluorescent signal generation.ResultsWe applied an experimental design approach for the optimization of the antibody concentrations used in the sandwich ELISA. The assay sensitivity was 0.1 ng of C-P4H. The method was utilized for the analysis of C-P4H accumulation in crude cell extracts of E. coli overexpressing C-P4H. The sandwich ELISA signals obtained demonstrated a very good correlation with the detected protein activity levels measured with the standard radioactive assay. The developed assay was applied to optimize C-P4H production in E. coli Origami in a system where the C-P4H subunits expression acted under control by different promoters. The experiments performed in a shake flask fed-batch system (EnBase) verified earlier observations that cell density and oxygen supply are critical factors for the use of the inducer anhydrotetracycline and thus for the soluble C-P4H yield.ConclusionsHere we show an example of sandwich ELISA usage for quantifying multimeric proteins. The method was developed for monitoring the amount of recombinant C-P4H tetramer in crude E. coli extracts. Due to the specificity of the antibodies used in the assay against the different C-P4H subunits, the method detects the entire holoenzyme, and the signal is not disturbed by background expression of the separate subunits.

Dataset Information

Structure and Mechanism of a Viral Collagen Prolyl Hydroxylase.

Publications

Structure and Mechanism of a Viral Collagen Prolyl Hydroxylase.

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