Joo, Sang Hoon Pemble, Charles W. Yang, Eun Gyeong Raetz, Christian R. H. Chung, Hak Suk 2024-01-19T21:34:24Z 2024-01-19T21:34:24Z 2021-09-04 2018-10 0022-2836 https://pubs.kist.re.kr/handle/201004/120871 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)alpha-ketoglutarate (alpha-KG)/O-2-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 (Kdo(MI)), from Methylacidiphilum infernorum V4. The de novo structure of Kdo(MI) apoprotein indicates that KdoO(MI) consists of 13 alpha helices and 11 beta strands, and has the jelly roll fold containing a metal binding motif, HXDX111H. Structures of Kdo(MI) bound to Co(II), Kdo(MI) bound to alpha-KG and Fe(III), and Kdo(MI) 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 alpha-KG. It was also observed that His225 is adjacent to the active site and plays an important role in the catalysis of KdoO(MI) without affecting substrate binding, possibly being involved in oxygen activation. The crystal structure of KdoO(MI) 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. (C) 2018 Elsevier Ltd. All rights reserved. English ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD Biochemical and Structural Insights into an Fe (II)/alpha-Ketoglutarate/O-2-Dependent Dioxygenase, Kdo 3-Hydroxylase (KdoO) Article 10.1016/j.jmb.2018.07.029 1 JOURNAL OF MOLECULAR BIOLOGY, v.430, no.21, pp.4036 - 4048 JOURNAL OF MOLECULAR BIOLOGY 430 21 4036 4048 N scie scopus 000448493400012 2-s2.0-85051650296 Biochemistry & Molecular Biology Biochemistry & Molecular Biology Article OXIDATION ALIGNMENT ESCHERICHIA-COLI LIPOPOLYSACCHARIDE MECHANISM Kdo 3-hydroxylase (KdoO) x-ray structure Fe(II)/alpha-ketoglutarate/O-2-dependent dioxygenase lipopolysaccharide biosynthesis deoxysugar oxidase