Apo structure of the Kdo hydroxylase KdoO, a non-heme Fe(II) alphaketoglutarate dependent dioxygenase

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)/α-ketoglutarate (α-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 α helices and 11 β strands, and has the jelly roll fold containing a metal binding motif, HXDX111H. Structures of KdoMI bound to Co(II), KdoMI bound to α-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 α-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.

[1]  C. Schofield,et al.  2-Oxoglutarate-Dependent Oxygenases. , 2018, Annual review of biochemistry.

[2]  I. Landrieu,et al.  Structural basis for oxygen degradation domain selectivity of the HIF prolyl hydroxylases , 2016, Nature Communications.

[3]  R. Hausinger,et al.  Catalytic Mechanisms of Fe(II)- and 2-Oxoglutarate-dependent Oxygenases* , 2015, The Journal of Biological Chemistry.

[4]  E. Yang,et al.  Kdo hydroxylase is an inner core assembly enzyme in the Ko-containing lipopolysaccharide biosynthesis. , 2014, Biochemical and biophysical research communications.

[5]  C. Raetz,et al.  Dioxygenases in Burkholderia ambifaria and Yersinia pestis that hydroxylate the outer Kdo unit of lipopolysaccharide , 2010, Proceedings of the National Academy of Sciences.

[6]  C. Raetz,et al.  Interchangeable domains in the Kdo transferases of Escherichia coli and Haemophilus influenzae. , 2010, Biochemistry.

[7]  Liisa Holm,et al.  Dali server: conservation mapping in 3D , 2010, Nucleic Acids Res..

[8]  George M. Sheldrick,et al.  Experimental phasing with SHELXC/D/E: combining chain tracing with density modification , 2010, Acta crystallographica. Section D, Biological crystallography.

[9]  Vincent B. Chen,et al.  MolProbity: all-atom structure validation for macromolecular crystallography , 2009, Acta crystallographica. Section D, Biological crystallography.

[10]  Hayyoung Lee,et al.  The structural basis of lipopolysaccharide recognition by the TLR4–MD-2 complex , 2009, Nature.

[11]  C. Raetz,et al.  Steady-state kinetics and mechanism of LpxD, the N-acyltransferase of lipid A biosynthesis. , 2008, Biochemistry.

[12]  M. Alam,et al.  Methane oxidation by an extremely acidophilic bacterium of the phylum Verrucomicrobia , 2007, Nature.

[13]  Randy J. Read,et al.  Phaser crystallographic software , 2007, Journal of applied crystallography.

[14]  Richa Agarwala,et al.  COBALT: constraint-based alignment tool for multiple protein sequences , 2007, Bioinform..

[15]  K. Colabroy,et al.  Structural studies on 3-hydroxyanthranilate-3,4-dioxygenase: the catalytic mechanism of a complex oxidation involved in NAD biosynthesis. , 2005, Biochemistry.

[16]  T. Bugg,et al.  Acid-base catalysis in the extradiol catechol dioxygenase reaction mechanism: site-directed mutagenesis of His-115 and His-179 in Escherichia coli 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB). , 2004, Biochemistry.

[17]  Michael I. Wilson,et al.  C. elegans EGL-9 and Mammalian Homologs Define a Family of Dioxygenases that Regulate HIF by Prolyl Hydroxylation , 2001, Cell.

[18]  Nathan A. Baker,et al.  Electrostatics of nanosystems: Application to microtubules and the ribosome , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[19]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[20]  C Combet,et al.  NPS@: network protein sequence analysis. , 2000, Trends in biochemical sciences.

[21]  Thomas L. Madden,et al.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. , 1997, Nucleic acids research.

[22]  O. Holst,et al.  Deletion of the heptosyltransferase genes rfaC and rfaF in Escherichia coli K-12 results in an Re-type lipopolysaccharide with a high degree of 2-aminoethanol phosphate substitution. , 1997, European journal of biochemistry.

[23]  J. Walker,et al.  Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. , 1996, Journal of molecular biology.

[24]  J. Thompson,et al.  CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. , 1994, Nucleic acids research.

[25]  W. J. Dyer,et al.  A rapid method of total lipid extraction and purification. , 1959, Canadian journal of biochemistry and physiology.

[26]  R. Dulbecco,et al.  PLAQUE FORMATION AND ISOLATION OF PURE LINES WITH POLIOMYELITIS VIRUSES , 1954, The Journal of experimental medicine.

[27]  W. Delano.  The PyMOL Molecular Graphics System , 2002 .

[28]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[29]  H. Nojima,et al.  High efficiency transformation of Escherichia coli with plasmids. , 1990, Gene.

[30]  Jeffrey H. Miller.  Experiments in molecular genetics , 1972 .