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

Abstract

Verrucomicrobium spinosum is a Gram-negative bacterium that is related to bacteria from the genus Chlamydia. The bacterium is pathogenic towards Drosophila melanogaster and Caenorhabditis elegans, using a type III secretion system to facilitate pathogenicity. V. spinosum 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 V. spinosum genome provides evidence that the bacterium should be able to synthesize peptidoglycan de novo, since all of the necessary genes are present. The enzyme UDP-N-acetylmuramoyl-l-alanyl-d-glutamate: meso-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 murE ortholog from V. spinosum (murE Vs) was cloned and was shown to possess UDP-MurNAc-l-Ala-d-Glu:meso-2,6-diaminopimelate ligase activity in vivo using functional complementation. In vitro analysis using the purified recombinant enzyme demonstrated that MurEVs has a pH optimum of 9.6 and a magnesium optimum of 30 mM. meso-Diaminopimelate was the preferred substrate with a K 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 MurEVs. Our kinetic analysis and structural model of MurEVs is consistent with other MurE enzymes from Gram-negative bacteria that have been characterized. To verify that V. spinosum incorporates diaminopimelate into its cell wall, we purified peptidoglycan from a V. spinosum culture; analysis revealed the presence of diaminopimelate, consistent with that of a bona fide peptidoglycan from Gram-negative bacteria.

Evolutionary Cell Biology of Division Mode in the Bacterial Planctomycetes-Verrucomicrobia- Chlamydiae Superphylum

“…PG had also been detected in some, but not all, verrucomicrobial species investigated (Yoon et al, 2007a,b, 2008a,b, 2010). In agreement, most PG synthesis genes were detected in Verrucomicrobium spinosum and the functionality of at least one of them has been confirmed (McGroty et al, 2013). Recently, PG has been detected in subdivision 5 of Verrucomicrobia (Spring et al, 2016).…”

Identification and Partial Characterization of a Novel UDP-N-Acetylenolpyruvoylglucosamine Reductase/UDP-N-Acetylmuramate:l-Alanine Ligase Fusion Enzyme from Verrucomicrobium spinosum DSM 4136T

“…Amino acids Gly and l -Ser, which are found at position 1 of the peptide stem in some bacteria (Schleifer and Kandler, 1972 ; Vollmer et al, 2008 ), were also tested as substrates (Table 4 ). l -Ala was the best substrate, which is in agreement with the amino acid composition of V. spinosum peptidoglycan (McGroty et al, 2013 ). However, the difference with the two others was not as important as for the E. coli MurC ortholog (Liger et al, 1995 ).…”

“…The phylum is considered to have two sister phyla, Chlamydiae and Lentisphaerae (Cho et al, 2004 ). Members of the Verrucomicrobia are of interest due to their close evolutionary relationship to bacteria from the genus Chlamydia in addition to their unusual morphology of possessing wart-like and tube-like appendages that protrude from the cell membrane, commonly referred to as prosthecae (Wagner and Horn, 2006 ; McGroty et al, 2013 ). Most of the research that has been done with bacteria from this phylum has been done using Verrucomicrobium spinosum as the model organism.…”

“…The bacterium was found to employ the type III secretion system and is pathogenic toward Drosophila melanogaster and Caenorhabditis elegans (Sait et al, 2011 ). In addition, research from our group recently demonstrated that the bacterium employs the L,L-diaminopimelate aminotransferase (DapL) pathway for the synthesis of meso- diaminopimelate involved both in the cross-linking of peptidoglycan (PG) and in lysine anabolism (Nachar et al, 2012 ; McGroty et al, 2013 ). Due to the morphological complexity and unusual cellular plan of V. spinosum , the synthesis of PG is of interest to our group given its close relationship to the pathogenic organisms from the genus Chlamydia.…”

The purification, crystallization and preliminary X-ray diffraction analysis of two isoforms ofmeso-diaminopimelate decarboxylase fromArabidopsis thaliana

“…In addition, many bacteria use lysine, or its precursor diaminopimelate, as a component of their peptidoglycan cell wall, which is necessary for maintaining the structural integrity of the cell (Dogovski et al, 2012;McGroty et al, 2013). Animals do not possess the enzymes for synthesizing lysine de novo and must obtain it through their diet, while plants and bacteria rely on their own production of the amino acid for growth (Jander & Joshi, 2009).…”