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

Kubra F Naqvi,Han Ming Gan,Delphine Patin,Hélène Barreteau,André O Hudson,Matthew S Wheatley,Didier Blanot,Dominique Mengin-Lecreulx,Michael A Savka,Renwick C J Dobson

doi:10.3389/fmicb.2016.00362

Abstract

The enzymes involved in synthesizing the bacterial cell wall are attractive targets for the design of antibacterial compounds, since this pathway is essential for bacteria and is absent in animals, particularly humans. A survey of the genome of a bacterium that belongs to the phylum Verrucomicrobia, the closest free-living relative to bacteria from the Chlamydiales phylum, shows genetic evidence that Verrucomicrobium spinosum possesses a novel fusion open reading frame (ORF) annotated by the locus tag (VspiD_010100018130). The ORF, which is predicted to encode the enzymes UDP-N-acetylenolpyruvoylglucosamine reductase (MurB) and UDP-N-acetylmuramate:l-alanine ligase (MurC) that are involved in the cytoplasmic steps of peptidoglycan biosynthesis, was cloned. In vivo analyses using functional complementation showed that the fusion gene was able to complement Escherichia coli murB and murC temperature sensitive mutants. The purified recombinant fusion enzyme (MurB/CVs) was shown to be endowed with UDP-N-acetylmuramate:l-alanine ligase activity. In vitro analyses demonstrated that the latter enzyme had a pH optimum of 9.0, a magnesium optimum of 10 mM and a temperature optimum of 44–46°C. Its apparent Km values for ATP, UDP-MurNAc, and l-alanine were 470, 90, and 25 μM, respectively. However, all attempts to demonstrate an in vitro UDP-N-acetylenolpyruvoylglucosamine reductase (MurB) activity were unsuccessful. Lastly, Hidden Markov Model-based similarity search and phylogenetic analysis revealed that this fusion enzyme could only be identified in specific lineages within the Verrucomicrobia phylum.

Highlights

Bacteria belonging to the Verrucomicrobia phylum are Gramnegative heterotrophic organisms that are generally found in soil and fresh water environments
We were interested to assess if the fusion enzyme had the domains that are indicative of typical MurB and MurC enzymes
This analysis demonstrated that the residues responsible for the MurC activity were located toward the Nterminal end of the fusion enzyme, while those for the MurB activity are located toward the C-terminal end

Summary

Introduction

Bacteria belonging to the Verrucomicrobia phylum are Gramnegative heterotrophic organisms that are generally found in soil and fresh water environments. 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). 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 recent discovery of PG in Chlamydia has made this project more intriguing, given the fact that even though βlactam antibiotics are effective against Chlamydia, definitive evidence of PG in Chlamydial species has been lacking until this recent discovery (Pilhofer et al, 2013; Packiam et al, 2015)

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Results

Conclusion