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

다국어 초록 (Multilingual Abstract)

<P><B>Abstract</B></P> <P>During lipopolysaccharide biosynthesis in several pathogens, including <I>Burkholderia</I> and <I>Yersinia</I>, 3-deoxy-<SMALL>D</SMALL>-<I>manno</I>-oct-2-ulosonic acid (Kdo) 3-hydroxylase, otherwise referred to as KdoO, converts Kdo to <SMALL>D</SMALL>-glycero-<SMALL>D</SMALL>-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 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<SUB>MI</SUB>), from <I>Methylacidiphilum infernorum V4</I>. The <I>de novo</I> structure of Kdo<SUB>MI</SUB> apoprotein indicates that KdoO<SUB>MI</SUB> consists of 13 α helices and 11 β strands, and has the jelly roll fold containing a metal binding motif, HXDX<SUB>111</SUB>H. Structures of Kdo<SUB>MI</SUB> bound to Co(II), Kdo<SUB>MI</SUB> bound to α-KG and Fe(III), and Kdo<SUB>MI</SUB> 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 α-KG. It was also observed that His225 is adjacent to the active site and plays an important role in the catalysis of KdoO<SUB>MI</SUB> without affecting substrate binding, possibly being involved in oxygen activation. The crystal structure of KdoO<SUB>MI</SUB> 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.</P> <P><B>Highlights</B></P> <P> <UL> <LI> KdoO converts Kdo to Ko during LPS biosynthesis. </LI> <LI> Minf_1012 from <I>Methylacidiphilum infernorum</I> functions as KdoO<SUB>MI</SUB>. </LI> <LI> The first completed structures of KdoO<SUB>MI</SUB> are determined at 1.45- to 1.94-Å resolution. </LI> <LI> The structure of KdoO<SUB>MI</SUB> reveals a metal binding motif HXDX<SUB> <I>n</I> > 40</SUB>H. </LI> <LI> Cosubstrate bound KdoO<SUB>MI</SUB> and mutagenesis study show important residues for catalysis. </LI> </UL> </P> <P><B>Graphical Abstract</B></P> <P>[DISPLAY OMISSION]</P>