Dataset Information

Biochemical and Structural Insights into an Fe(II)/?-Ketoglutarate/O2-Dependent Dioxygenase, Kdo 3-Hydroxylase (KdoO).

ABSTRACT: During lipopolysaccharide biosynthesis in several pathogens, including Burkholderia and Yersinia, 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) 3-hydroxylase, otherwise referred to as KdoO, converts Kdo to d-glycero-d-talo-oct-2-ulosonic acid (Ko) in an Fe(II)/?-ketoglutarate (?-KG)/O2-dependent manner. This conversion renders the bacterial outer membrane more stable and resistant to stresses such as an acidic environment. KdoO is a membrane-associated, deoxy-sugar hydroxylase that does not show significant sequence identity with any known enzymes, and its structural information has not been previously reported. Here, we report the biochemical and structural characterization of KdoO, Minf_1012 (KdoMI), from Methylacidiphilum infernorum V4. The de novo structure of KdoMI apoprotein indicates that KdoOMI consists of 13 ? helices and 11 ? strands, and has the jelly roll fold containing a metal binding motif, HXDX111H. Structures of KdoMI bound to Co(II), KdoMI bound to ?-KG and Fe(III), and KdoMI bound to succinate and Fe(III), in addition to mutagenesis analysis, indicate that His146, His260, and Asp148 play critical roles in Fe(II) binding, while Arg127, Arg162, Arg174, and Trp176 stabilize ?-KG. It was also observed that His225 is adjacent to the active site and plays an important role in the catalysis of KdoOMI without affecting substrate binding, possibly being involved in oxygen activation. The crystal structure of KdoOMI is the first completed structure of a deoxy-sugar hydroxylase, and the data presented here have provided mechanistic insights into deoxy-sugar hydroxylase, KdoO, and lipopolysaccharide biosynthesis.

SUBMITTER: Joo SH

PROVIDER: S-EPMC6186499 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Biochemical and Structural Insights into an Fe(II)/α-Ketoglutarate/O<sub>2</sub>-Dependent Dioxygenase, Kdo 3-Hydroxylase (KdoO).

Joo Sang Hoon SH Pemble Charles W CW Yang Eun Gyeong EG Raetz Christian R H CRH Chung Hak Suk HS

Journal of molecular biology 20180807 21

During lipopolysaccharide biosynthesis in several pathogens, including Burkholderia and Yersinia, 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) 3-hydroxylase, otherwise referred to as KdoO, converts Kdo to d-glycero-d-talo-oct-2-ulosonic acid (Ko) in an Fe(II)/α-ketoglutarate (α-KG)/O<sub>2</sub>-dependent manner. This conversion renders the bacterial outer membrane more stable and resistant to stresses such as an acidic environment. KdoO is a membrane-associated, deoxy-sugar hydroxylase that does n ...[more]

PMID: 30092253

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Project description:Acetylacetone dioxygenase (Dke1) is a bacterial enzyme that catalyzes the dioxygen-dependent degradation of β-dicarbonyl compounds. The Dke1 active site contains a nonheme monoiron(II) center facially ligated by three histidine residues (the 3His triad); coordination of the substrate in a bidentate manner provides a five-coordinate site for O(2) binding. Recently, we published the synthesis and characterization of a series of ferrous β-diketonato complexes that faithfully mimic the enzyme-substrate intermediate of Dke1 (Park, H.; Baus, J.S.; Lindeman, S.V.; Fiedler, A.T. Inorg. Chem.2011, 50, 11978-11989). The 3His triad was modeled with three different facially coordinating N3 supporting ligands, and substituted β-diketonates (acac(X)) with varying steric and electronic properties were employed. Here, we describe the reactivity of our Dke1 models toward O(2) and its surrogate nitric oxide (NO), and report the synthesis of three new Fe(II) complexes featuring the anions of dialkyl malonates. Exposure of [Fe((Me2)Tp)(acac(X))] complexes (where (R2)Tp = hydrotris(pyrazol-1-yl)borate with R-groups at the 3- and 5-positions of the pyrazole rings) to O(2) at -70 °C in toluene results in irreversible formation of green chromophores (λ(max) ∼750 nm) that decay at temperatures above -60 °C. Spectroscopic and computational analyses suggest that these intermediates contain a diiron(III) unit bridged by a trans μ-1,2-peroxo ligand. The green chromophore is not observed with analogous complexes featuring (Ph2)Tp and (Ph)TIP ligands (where (Ph)TIP = tris(2-phenylimidazoly-4-yl)phosphine), since the steric bulk of the phenyl substituents prevents formation of dinuclear species. While these complexes are largely inert toward O(2), (Ph2)Tp-based complexes with dialkyl malonate anions exhibit dioxygenase activity and thus serve as functional Dke1 models. The Fe/acac(X) complexes all react readily with NO to yield high-spin (S = 3/2) {FeNO}(7) adducts that were characterized with crystallographic, spectroscopic, and computational methods. Collectively, the results presented here enhance our understanding of the chemical factors involved in the oxidation of aliphatic substrates by nonheme iron dioxygenases.

Dataset Information

Biochemical and Structural Insights into an Fe(II)/?-Ketoglutarate/O2-Dependent Dioxygenase, Kdo 3-Hydroxylase (KdoO).

Publications

Biochemical and Structural Insights into an Fe(II)/α-Ketoglutarate/O<sub>2</sub>-Dependent Dioxygenase, Kdo 3-Hydroxylase (KdoO).

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