Activity and Crystal Structure of <i>Arabidopsis thaliana</i>UDP-<i>N</i>-Acetylglucosamine Acyltransferase

Joo, Sang Hoon,Chung, Hak Suk,Raetz, Christian R. H.,Garrett, Teresa A. American Chemical Society 2012 Biochemistry Vol.51 No.21

<P>The UDP-<I>N</I>-acetylglucosamine (UDP-GlcNAc) acyltransferase, encoded by <I>lpxA</I>, catalyzes the first step of lipid A biosynthesis in Gram-negative bacteria, the (<I>R</I>)-3-hydroxyacyl-ACP-dependent acylation of the 3-OH group of UDP-GlcNAc. Recently, we demonstrated that the <I>Arabidopsis thaliana</I> orthologs of six enzymes of the bacterial lipid A pathway produce lipid A precursors with structures similar to those of <I>Escherichia coli</I> lipid A precursors [Li, C., et al. (2011) <I>Proc. Natl. Acad. Sci. U.S.A. 108</I>, 11387–11392]. To build upon this finding, we have cloned, purified, and determined the crystal structure of the <I>A. thaliana</I> LpxA ortholog (AtLpxA) to 2.1 Å resolution. The overall structure of AtLpxA is very similar to that of <I>E. coli</I> LpxA (EcLpxA) with an α-helical-rich C-terminus and characteristic N-terminal left-handed parallel β-helix (LβH). All key catalytic and chain length-determining residues of EcLpxA are conserved in AtLpxA; however, AtLpxA has an additional coil and loop added to the LβH not seen in EcLpxA. Consistent with the similarities between the two structures, purified AtLpxA catalyzes the same reaction as EcLpxA. In addition, <I>A. thaliana lpxA</I> complements an <I>E. coli</I> mutant lacking the chromosomal <I>lpxA</I> and promotes the synthesis of lipid A in vivo similar to the lipid A produced in the presence of <I>E. coli lpxA</I>. This work shows that AtLpxA is a functional UDP-GlcNAc acyltransferase that is able to catalyze the same reaction as EcLpxA and supports the hypothesis that lipid A molecules are biosynthesized in <I>Arabidopsis</I> and other plants.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/bichaw/2012/bichaw.2012.51.issue-21/bi3002242/production/images/medium/bi-2012-002242_0007.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/bi3002242'>ACS Electronic Supporting Info</A></P>

Multi-site campaign for transit timing variations of WASP-12 b: possible detection of a long-period signal of planetary origin

Maciejewski, G.,Dimitrov, D.,Seeliger, M.,Raetz, St.,Bukowiecki, Ł.,Kitze, M.,Errmann, R.,Nowak, G.,Niedzielski, A.,Popov, V.,Marka, C.,Goź,dziewski, K.,Neuhä,user, R.,Ohlert, J.,Hinse, T. C. EDP Sciences 2013 Astronomy and astrophysics Vol.551 No.-

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

Joo, Sang Hoon,Pemble IV, Charles W.,Yang IV, Eun Gyeong,Raetz IV, Christian R.H.,Chung IV, Hak Suk Elsevier 2018 Journal of molecular biology Vol.430 No.21

<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>

Pathogenicity of <i>Yersinia pestis</i> Synthesis of 1-Dephosphorylated Lipid A

Sun, Wei,Six, David A.,Reynolds, C. Michael,Chung, Hak Suk,Raetz, Christian R. H.,Curtiss 3rd, Roy American Society for Microbiology 2013 Infection and immunity Vol.81 No.4

<P>Synthesis of <I>Escherichia coli</I> LpxL, which transfers a secondary laurate chain to the 2′ position of lipid A, in <I>Yersinia pestis</I> produced bisphosphoryl hexa-acylated lipid A at 37°C, leading to significant attenuation of virulence. Our previous observations also indicated that strain χ10015(pCD1Ap) (Δ<I>lpxP32</I>::P<SUB>lpxL</SUB> <I>lpxL</I>) stimulated a strong inflammatory reaction but sickened mice before recovery and retained virulence via intranasal (i.n.) infection. The development of live, attenuated <I>Y. pestis</I> vaccines may be facilitated by detoxification of its lipopolysaccharide (LPS). Heterologous expression of the lipid A 1-phosphatase, LpxE, from <I>Francisella tularensis</I> in <I>Y. pestis</I> yields predominantly 1-dephosphorylated lipid A, as confirmed by mass spectrometry. Results indicated that expression of LpxE on top of LpxL provided no significant reduction in virulence of <I>Y. pestis</I> in mice when it was administered i.n. but actually reduced the 50% lethal dose (LD<SUB>50</SUB>) by 3 orders of magnitude when the strain was administered subcutaneously (s.c.). Additionally, LpxE synthesis in wild-type <I>Y. pestis</I> KIM6+(pCD1Ap) led to slight attenuation by s.c. inoculation but no virulence change by i.n. inoculation in mice. In contrast to <I>Salmonella enterica</I>, expression of LpxE does not attenuate the virulence of <I>Y. pestis</I>.</P>

The Origin of 8-Amino-3,8-dideoxy-<small>d</small>-manno-octulosonic Acid (Kdo8N) in the Lipopolysaccharide of <i>Shewanella oneidensis</i>

Gattis, Samuel G.,Chung, Hak Suk,Trent, M. Stephen,Raetz, Christian R. H. American Society for Biochemistry and Molecular Bi 2013 The Journal of biological chemistry Vol.288 No.13

<P>Lipopolysaccharide (LPS; endotoxin) is an essential component of the outer monolayer of nearly all Gram-negative bacteria. LPS is composed of a hydrophobic anchor, known as lipid A, an inner core oligosaccharide, and a repeating O-antigen polysaccharide. In nearly all species, the first sugar bridging the hydrophobic lipid A and the polysaccharide domain is 3-deoxy-<SMALL>d</SMALL>-manno-octulosonic acid (Kdo), and thus it is critically important for LPS biosynthesis. Modifications to lipid A have been shown to be important for resistance to antimicrobial peptides as well as modulating recognition by the mammalian innate immune system. Therefore, lipid A derivatives have been used for development of vaccine strains and vaccine adjuvants. One derivative that has yet to be studied is 8-amino-3,8-dideoxy-<SMALL>d</SMALL>-manno-octulosonic acid (Kdo8N), which is found exclusively in marine bacteria of the genus <I>Shewanella</I>. Using bioinformatics, a candidate gene cluster for Kdo8N biosynthesis was identified in <I>Shewanella oneidensis</I>. Expression of these genes recombinantly in <I>Escherichia coli</I> resulted in lipid A containing Kdo8N, and <I>in vitro</I> assays confirmed their proposed enzymatic function. Both the <I>in vivo</I> and <I>in vitro</I> data were consistent with direct conversion of Kdo to Kdo8N prior to its incorporation into the Kdo8N-lipid A domain of LPS by a metal-dependent oxidase followed by a glutamate-dependent aminotransferase. To our knowledge, this oxidase is the first enzyme shown to oxidize an alcohol using a metal and molecular oxygen, not NAD(P)<SUP>+</SUP>. Creation of an <I>S. oneidensis</I> in-frame deletion strain showed increased sensitivity to the cationic antimicrobial peptide polymyxin as well as bile salts, suggesting a role in outer membrane integrity.</P>

Mutants Resistant to LpxC Inhibitors by Rebalancing Cellular Homeostasis

Zeng, Daina,Zhao, Jinshi,Chung, Hak Suk,Guan, Ziqiang,Raetz, Christian R. H.,Zhou, Pei American Society for Biochemistry and Molecular Bi 2013 The Journal of biological chemistry Vol.288 No.8