title: Structure and function of the DUF2233 domain in bacteria and in the human mannose 6-phosphate uncovering enzyme.
abstract: DUF2233, a domain of unknown function (DUF), is present in many bacterial and several viral proteins and was also identified in the mammalian transmembrane glycoprotein N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase ("uncovering enzyme" (UCE)). We report the crystal structure of BACOVA_00430, a 315-residue protein from the human gut bacterium Bacteroides ovatus that is the first structural representative of the DUF2233 protein family. A notable feature of this structure is the presence of a surface cavity that is populated by residues that are highly conserved across the entire family. The crystal structure was used to model the luminal portion of human UCE (hUCE), which is involved in targeting of lysosomal enzymes. Mutational analysis of several residues in a highly conserved surface cavity of hUCE revealed that they are essential for function. The bacterial enzyme (BACOVA_00430) has ∼1% of the catalytic activity of hUCE toward the substrate GlcNAc-P-mannose, the precursor of the Man-6-P lysosomal targeting signal. GlcNAc-1-P is a poor substrate for both enzymes. We conclude that, for at least a subset of proteins in this family, DUF2233 functions as a phosphodiester glycosidase.
authors.affiliation: Joint Center for Structural Genomics, Menlo Park, California 94025; Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, California 94025.
authors.forename: Debanu
authors.initials: D
authors.lastname: Das
authors.affiliation: Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: Wang-Sik
authors.initials: WS
authors.lastname: Lee
authors.affiliation: Joint Center for Structural Genomics, Menlo Park, California 94025; Protein Sciences Department, Genomics Institute of the Novartis Research Foundation, San Diego, California 92121.
authors.forename: Joanna C
authors.initials: JC
authors.lastname: Grant
authors.affiliation: Joint Center for Structural Genomics, Menlo Park, California 94025; Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, California 94025.
authors.forename: Hsiu-Ju
authors.initials: HJ
authors.lastname: Chiu
authors.affiliation: Joint Center for Structural Genomics, Menlo Park, California 94025; Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California 92037.
authors.forename: Carol L
authors.initials: CL
authors.lastname: Farr
authors.affiliation: Joint Center for Structural Genomics, Menlo Park, California 94025; Protein Sciences Department, Genomics Institute of the Novartis Research Foundation, San Diego, California 92121.
authors.forename: Julie
authors.initials: J
authors.lastname: Vance
authors.affiliation: Joint Center for Structural Genomics, Menlo Park, California 94025; Protein Sciences Department, Genomics Institute of the Novartis Research Foundation, San Diego, California 92121.
authors.forename: Heath E
authors.initials: HE
authors.lastname: Klock
authors.affiliation: Joint Center for Structural Genomics, Menlo Park, California 94025; Protein Sciences Department, Genomics Institute of the Novartis Research Foundation, San Diego, California 92121.
authors.forename: Mark W
authors.initials: MW
authors.lastname: Knuth
authors.affiliation: Joint Center for Structural Genomics, Menlo Park, California 94025; Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, California 94025.
authors.forename: Mitchell D
authors.initials: MD
authors.lastname: Miller
authors.affiliation: Joint Center for Structural Genomics, Menlo Park, California 94025; Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California 92037.
authors.forename: Marc-André
authors.initials: MA
authors.lastname: Elsliger
authors.affiliation: Joint Center for Structural Genomics, Menlo Park, California 94025; Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, California 94025.
authors.forename: Ashley M
authors.initials: AM
authors.lastname: Deacon
authors.affiliation: Joint Center for Structural Genomics, Menlo Park, California 94025; Program on Bioinformatics and Systems Biology, Sanford-Burnham Medical Research Institute, La Jolla, California 92037; Center for Research in Biological Systems, University of California San Diego, La Jolla, California 92093.
authors.forename: Adam
authors.initials: A
authors.lastname: Godzik
authors.affiliation: Joint Center for Structural Genomics, Menlo Park, California 94025; Protein Sciences Department, Genomics Institute of the Novartis Research Foundation, San Diego, California 92121; Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California 92037.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Lesley
authors.affiliation: Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110. Electronic address: skornfel@dom.wustl.edu.
authors.forename: Stuart
authors.initials: S
authors.lastname: Kornfeld
authors.affiliation: Joint Center for Structural Genomics, Menlo Park, California 94025; Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California 92037. Electronic address: wilson@scripps.edu.
authors.forename: Ian A
authors.initials: IA
authors.lastname: Wilson
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001439:Bacteroides / Q000201:enzymology*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006801:Humans; D016296:Mutagenesis; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D040681:Structural Homology, Protein
chemical_list: D001426:Bacterial Proteins; D010727:Phosphoric Diester Hydrolases; C026645:N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase
keywords: Crystal Structure; DUF2233; Functional Genomics; Genomics; Glycoprotein; Structural Biology; Structural Genomics; Uncovering Enzyme