title: The role of histidine 200 in MndD, the Mn(II)-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenase from Arthrobacter globiformis CM-2, a site-directed mutagenesis study.
abstract: The manganese-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenase (MndD) from Arthrobacter globiformis CM-2 is an extradiol-cleaving catechol dioxygenase that catalyzes aromatic ring cleavage of 3,4-dihydroxyphenylacetate (DHPA). Based on the recent crystal structure of the MndD-DHPA complex, a series of site-directed mutations were made at a conserved second-sphere residue, histidine 200, to gain insight into and clarify the role this residue plays in the Mn(II)-dependent catalytic mechanism. In this study, we report the activities and spectroscopic data of these H200 variants and their DHPA and 4-nitrocatechol (4-NC) complexes. The data collected from wild-type and mutant MndDs are consistent with a role for H200 interacting with a manganese-bound dioxygen moiety and are inconsistent with other previously proposed roles involving proton transfer. Spectroscopic observations, including unique low-field EPR signals found when DHPA and 4-NC are bound to the Mn(II) center of MndD, are discussed and their relationship to dioxygen activation catalyzed in MndD is explored.
authors.affiliation: Department of Chemistry and Center for Metals in Biocatalysis, University of Minnesota, 207 Pleasant St. SE, Minneapolis, MN 55455, USA.
authors.forename: Joseph P
authors.initials: JP
authors.lastname: Emerson
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Wagner
authors.forename: Mark F
authors.initials: MF
authors.lastname: Reynolds
authors.forename: Lawrence
authors.initials: L
authors.lastname: Que
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Sadowsky
authors.forename: Lawrence P
authors.initials: LP
authors.lastname: Wackett
mesh_terms: D001173:Arthrobacter / Q000201:enzymology* / Q000235:genetics*; D002396:Catechols / Q000737:chemistry; D018360:Crystallography, X-Ray; D049308:Dioxygenases / Q000737:chemistry* / Q000235:genetics*; D004578:Electron Spin Resonance Spectroscopy; D006639:Histidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008345:Manganese / Q000737:chemistry*; D008670:Metals / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation
chemical_list: D002396:Catechols; D008670:Metals; D008345:Manganese; D006639:Histidine; D049308:Dioxygenases; C020055:3,4-dihydroxyphenylacetate 2,3-dioxygenase; C001833:4-nitrocatechol