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      Crystal structures of Phanerochaete chrysosporium pyranose 2-oxidase suggest that the N-terminus acts as a propeptide that assists in homotetramer assembly

      research-article
      a , b , a , c , c , c , c , a , b , *
      FEBS Open Bio
      Elsevier
      Pyranose 2-oxidase, Propeptide, Oligomerization, Thermostability, Crystal structure, Lignin degradation, 2FGlc, 2-deoxy-2-fluoro-d-glucose, 3FGlc, 3-deoxy-3-fluoro-d-glucose, DTT, dithiothreitol, HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, IMAC, by immobilized metal ion affinity chromatography, IPTG, β-d-1-thiogalactopyranoside, MES, 2-(N-morpholino) ethanesulfonic acid, MWCO, molecular weight cut off, P2O, pyranose oxidase, PBS, phosphate buffered saline, PDB, Protein Data Bank, PEG, polyethylene glycol, TEV, Tobacco Etch Virus

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          Abstract

          The flavin-dependent homotetrameric enzyme pyranose 2-oxidase (P2O) is found mostly, but not exclusively, in lignocellulose-degrading fungi where it catalyzes the oxidation of β- d-glucose to the corresponding 2-keto sugar concomitantly with hydrogen peroxide formation during lignin solubilization. Here, we present crystal structures of P2O from the efficient lignocellulolytic basidiomycete Phanerochaete chrysosporium. Structures were determined of wild-type PcP2O from the natural fungal source, and two variants of recombinant full-length PcP2O, both in complex with the slow substrate 3-deoxy-3-fluoro- β- d-glucose. The active sites in PcP2O and P2O from Trametes multicolor ( TmP2O) are highly conserved with identical substrate binding. Our structural analysis suggests that the 17 °C higher melting temperature of PcP2O compared to TmP2O is due to an increased number of intersubunit salt bridges. The structure of recombinant PcP2O expressed with its natural N-terminal sequence, including a proposed propeptide segment, reveals that the first five residues of the propeptide intercalate at the interface between A and B subunits to form stabilizing, mainly hydrophobic, interactions. In the structure of mature PcP2O purified from the natural source, the propeptide segment in subunit A has been replaced by a nearby loop in the B subunit. We propose that the propeptide in subunit A stabilizes the A/B interface of essential dimers in the homotetramer and that, upon maturation, it is replaced by the loop in the B subunit to form the mature subunit interface. This would imply that the propeptide segment of PcP2O acts as an intramolecular chaperone for oligomerization at the A/B interface of the essential dimer.

          Highlights

          • Structures of pyranose 2-oxidase from Phanerochaete chrysosporium were determined.

          • The N-terminus may act as a propeptide with a role in homotetramer assembly.

          • A large number of salt bridges between subunits provides thermostability.

          • The substrate is bound in the productive binding mode for oxidation at C2.

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          Most cited references47

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          Linking crystallographic model and data quality.

          In macromolecular x-ray crystallography, refinement R values measure the agreement between observed and calculated data. Analogously, R(merge) values reporting on the agreement between multiple measurements of a given reflection are used to assess data quality. Here, we show that despite their widespread use, R(merge) values are poorly suited for determining the high-resolution limit and that current standard protocols discard much useful data. We introduce a statistic that estimates the correlation of an observed data set with the underlying (not measurable) true signal; this quantity, CC*, provides a single statistically valid guide for deciding which data are useful. CC* also can be used to assess model and data quality on the same scale, and this reveals when data quality is limiting model improvement.
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            Improved methods for building protein models in electron density maps and the location of errors in these models.

            Map interpretation remains a critical step in solving the structure of a macromolecule. Errors introduced at this early stage may persist throughout crystallographic refinement and result in an incorrect structure. The normally quoted crystallographic residual is often a poor description for the quality of the model. Strategies and tools are described that help to alleviate this problem. These simplify the model-building process, quantify the goodness of fit of the model on a per-residue basis and locate possible errors in peptide and side-chain conformations.
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              Optimal description of a protein structure in terms of multiple groups undergoing TLS motion.

              A single protein crystal structure contains information about dynamic properties of the protein as well as providing a static view of one three-dimensional conformation. This additional information is to be found in the distribution of observed electron density about the mean position of each atom. It is general practice to account for this by refining a separate atomic displacement parameter (ADP) for each atomic center. However, these same displacements are often described well by simpler models based on TLS (translation/libration/screw) rigid-body motion of large groups of atoms, for example interdomain hinge motion. A procedure, TLSMD, has been developed that analyzes the distribution of ADPs in a previously refined protein crystal structure in order to generate optimal multi-group TLS descriptions of the constituent protein chains. TLSMD is applicable to crystal structures at any resolution. The models generated by TLSMD analysis can significantly improve the standard crystallographic residuals R and R(free) and can reveal intrinsic dynamic properties of the protein.
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                Author and article information

                Contributors
                Journal
                FEBS Open Bio
                FEBS Open Bio
                FEBS Open Bio
                Elsevier
                2211-5463
                5 November 2013
                5 November 2013
                2013
                : 3
                : 496-504
                Affiliations
                [a ]KTH Royal Institute of Technology, School of Biotechnology, Albanova University Center, Roslagstullsbacken 21, S-10691 StockholmSweden
                [b ]Karolinska Institute, Department of Medical Biochemistry and Biophysics, Scheelelaboratoriet, Scheeles väg 2, S-17177 StockholmSweden
                [c ]BOKU University of Natural Resources and Life Sciences, Food Biotechnology Laboratory, A-1190 ViennaAustria
                Author notes
                [* ]Corresponding author at: KTH Royal Institute of Technology, School of Biotechnology, Albanova University Center, Roslagstullsbacken 21, S-10691StockholmSweden. Tel.: +46 8 55378296; fax: +46 8 55378468. divne@ 123456biotech.kth.se
                Article
                S2211-5463(13)00067-3
                10.1016/j.fob.2013.10.010
                3839853
                24282677
                66be3aeb-cc85-4fdd-9324-6357ed2ae765
                © 2013 The Authors

                This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 28 October 2013
                : 30 October 2013
                : 31 October 2013
                Categories
                Article

                pyranose 2-oxidase,propeptide,oligomerization,thermostability,crystal structure,lignin degradation,2fglc, 2-deoxy-2-fluoro-d-glucose,3fglc, 3-deoxy-3-fluoro-d-glucose,dtt, dithiothreitol,hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid,imac, by immobilized metal ion affinity chromatography,iptg, β-d-1-thiogalactopyranoside,mes, 2-(n-morpholino) ethanesulfonic acid,mwco, molecular weight cut off,p2o, pyranose oxidase,pbs, phosphate buffered saline,pdb, protein data bank,peg, polyethylene glycol,tev, tobacco etch virus

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