Biochemical Characterization of UDP-<i>N</i>-acetylmuramoyl-L-alanyl-D-glutamate: <i>meso</i>-2,6-diaminopimelate ligase (MurE) from <i>Verrucomicrobium spinosum</i> DSM 4136^T

<div><p><i>Verrucomicrobium spinosum</i> is a Gram-negative bacterium that is related to bacteria from the genus <i>Chlamydia</i>. The bacterium is pathogenic towards <i>Drosophila melanogaster</i> and <i>Caenorhabditis elegans</i>, using a type III secretion system to facilitate pathogenicity. <i>V. spinosum</i> employs the recently discovered l,l-diaminopimelate aminotransferase biosynthetic pathway to generate the bacterial cell wall and protein precursors diaminopimelate and lysine. A survey of the <i>V. spinosum</i> genome provides evidence that the bacterium should be able to synthesize peptidoglycan <i>de novo</i>, since all of the necessary genes are present. The enzyme UDP-<i>N</i>-acetylmuramoyl-l-alanyl-d-glutamate: <i>meso</i>-2,6-diaminopimelate ligase (MurE) (E.C. 6.3.2.15) catalyzes a reaction in the cytoplasmic step of peptidoglycan biosynthesis by adding the third amino acid residue to the peptide stem. The <i>murE</i> ortholog from <i>V. spinosum</i> (<i>murE</i>_Vs) was cloned and was shown to possess UDP-MurNAc-l-Ala-d-Glu:<i>meso</i>-2,6-diaminopimelate ligase activity <i>in vivo</i> using functional complementation. <i>In vitro</i> analysis using the purified recombinant enzyme demonstrated that MurE_Vs has a pH optimum of 9.6 and a magnesium optimum of 30 mM. <i>meso</i>-Diaminopimelate was the preferred substrate with a <i>K</i>_m of 17 µM, when compared to other substrates that are structurally related. Sequence alignment and structural analysis using homology modeling suggest that key residues that make up the active site of the enzyme are conserved in MurE_Vs. Our kinetic analysis and structural model of MurE_Vs is consistent with other MurE enzymes from Gram-negative bacteria that have been characterized. To verify that <i>V. spinosum</i> incorporates diaminopimelate into its cell wall, we purified peptidoglycan from a <i>V. spinosum</i> culture; analysis revealed the presence of diaminopimelate, consistent with that of a bona fide peptidoglycan from Gram-negative bacteria.</p></div

The monomer unit of the peptidoglycan structure.

<p>The disaccharide moiety is composed of the amino sugars <i>N</i>-acetylglucosamine (GlcNAc) and <i>N</i>-acetylmuramic (MurNAc) linked via a β-1,4 glycosidic bond. The amino acid at position 3 of the stem peptide is <i>meso</i>-diaminopimelic acid (R =  COOH) in most Gram-negative bacteria and l-lysine (R = H) in most Gram-positive bacteria.</p

Homolgy model of MurE_Vs.

<p>(a) The homology model of MurE_Vs highlighting domains A (grey), B (violet) and C (pink). (b) Shows the structure model of MurE_Vs bound to UDP-MurNAc-tripeptide (UMT) product (yellow). (c) Active site residue hypothesized to bind to UMT product is shown in red. The structure has been rotated 90° on the right panel for the better viewing of the binding pocket. (d) Cross eye stereo view showing the interaction between amino acid residues of the binding site and UMT product.</p

Expression and purification of recombinant MurE_Vs using His-tag affinity chromatography.

<p>Lane (1) protein makers (kDa); Lane (2) 10 µg of soluble protein from uninduced cells; Lane (3) 10 µg of soluble protein from induced cells; Lane (4) 1 µg of purified recombinant MurE_Vs. The proteins were resolved on 10% (w/v) acrylamide gel and were stained using Coomassie blue.</p

Analysis of crude and purified PG from <i>V. spinosum</i> DSM 4136^T.

a<p>Crude and purified PG designate the macromolecule before and after, respectively, treatment with pancreatin, pronase and trypsin (see Materials and Methods).</p

List of genes involved in PG metabolism of <i>V. spinosum</i> DSM 4136^T.

<p>The annotated gene product names are from NCBI (<a href="http://www.ncbi.nlm.nih.gov/protein/" target="_blank">www.ncbi.nlm.nih.gov/protein/</a>) queried of February 28, 2013. The pencillin-binding proteins (PBP) class designations are denoted by activity based on <b><u>p</u></b>rotein <b><u>fam</u></b>ily (pfam) domains. Class A and class B PBPs are high-molecular mass PBPs while class C PBPs are low-molecular mass PBPs. Class A PBPs are predicted to have both transglycosylase and transpeptidase activities; class B PBPs are predicted to have only transpeptidase activity; class C PBPs are predicted to have d,d-carboxypeptidase activity.</p

Multiple amino acid sequence alignment of five representative sequences of MurE.

<p>The residues that are predicted to be involved in binding in the active site are marked with a star below the sequence. The sequence identity score against MurE from <i>V. spinosum</i> was: <i>C. trachomatis,</i> 37%; <i>E. coli,</i> 35%; <i>P. carotovorum</i>, 36%; and <i>M. tuberculosis</i>. The multiple amino acid sequence alignment figure was generated using the ESPript 2.2 server (<a href="http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi" target="_blank">http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi</a>).</p