title: 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 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. 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.
authors.affiliation: Centre for Synthetic Biology of Fine and Specialty Chemicals (SYNBIOCHEM), Manchester Institute of Biotechnology, The University of Manchester , Manchester M1 7DN, United Kingdom.
authors.forename: James E
authors.initials: JE
authors.lastname: Longbotham
authors.affiliation: Centre for Synthetic Biology of Fine and Specialty Chemicals (SYNBIOCHEM), Manchester Institute of Biotechnology, The University of Manchester , Manchester M1 7DN, United Kingdom.
authors.forename: Colin
authors.initials: C
authors.lastname: Levy
authors.affiliation: Centre for Synthetic Biology of Fine and Specialty Chemicals (SYNBIOCHEM), Manchester Institute of Biotechnology, The University of Manchester , Manchester M1 7DN, United Kingdom.
authors.forename: Linus O
authors.initials: LO
authors.lastname: Johannissen
authors.affiliation: Chemistry Research Laboratory, University of Oxford , 12 Mansfield Road, Oxford OX1 3TA, United Kingdom.
authors.forename: Hanna
authors.initials: H
authors.lastname: Tarhonskaya
authors.affiliation: Chemistry Research Laboratory, University of Oxford , 12 Mansfield Road, Oxford OX1 3TA, United Kingdom.
authors.forename: Shuo
authors.initials: S
authors.lastname: Jiang
authors.affiliation: Chemistry Research Laboratory, University of Oxford , 12 Mansfield Road, Oxford OX1 3TA, United Kingdom.
authors.forename: Christoph
authors.initials: C
authors.lastname: Loenarz
authors.affiliation: Chemistry Research Laboratory, University of Oxford , 12 Mansfield Road, Oxford OX1 3TA, United Kingdom.
authors.forename: Emily
authors.initials: E
authors.lastname: Flashman
authors.affiliation: Centre for Synthetic Biology of Fine and Specialty Chemicals (SYNBIOCHEM), Manchester Institute of Biotechnology, The University of Manchester , Manchester M1 7DN, United Kingdom.
authors.forename: Sam
authors.initials: S
authors.lastname: Hay
authors.affiliation: Chemistry Research Laboratory, University of Oxford , 12 Mansfield Road, Oxford OX1 3TA, United Kingdom.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Schofield
authors.affiliation: Centre for Synthetic Biology of Fine and Specialty Chemicals (SYNBIOCHEM), Manchester Institute of Biotechnology, The University of Manchester , Manchester M1 7DN, United Kingdom.
authors.forename: Nigel S
authors.initials: NS
authors.lastname: Scrutton
mesh_terms: D020816:Amino Acid Motifs; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D007501:Iron / Q000737:chemistry*; D017862:Phycodnaviridae / Q000201:enzymology*; D064828:Prolyl Hydroxylases / Q000737:chemistry*; D014764:Viral Proteins / Q000737:chemistry*
chemical_list: D014764:Viral Proteins; D007501:Iron; D064828:Prolyl Hydroxylases