title: 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).
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/C Vs ) 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 K m 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.
authors.affiliation: Thomas H. Gosnell School of Life Sciences, Rochester Institute of Technology Rochester, NY, USA.
authors.forename: Kubra F
authors.initials: KF
authors.lastname: Naqvi
authors.affiliation: Institute for Integrative Biology of the Cell, CEA, CNRS, Univ Paris-Sud, Université Paris-Saclay Orsay, France.
authors.forename: Delphine
authors.initials: D
authors.lastname: Patin
authors.affiliation: Thomas H. Gosnell School of Life Sciences, Rochester Institute of Technology Rochester, NY, USA.
authors.forename: Matthew S
authors.initials: MS
authors.lastname: Wheatley
authors.affiliation: Thomas H. Gosnell School of Life Sciences, Rochester Institute of Technology Rochester, NY, USA.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Savka
authors.affiliation: Biomolecular Interaction Centre, School of Biological Sciences, University of CanterburyChristchurch, New Zealand; Department of Biochemistry and Molecular Biology, Bio21 Molecular and Biotechnology Institute, The University of MelbourneParkville, VIC, Australia.
authors.forename: Renwick C J
authors.initials: RC
authors.lastname: Dobson
authors.affiliation: Monash University Malaysia Genomics Facility, Monash University MalaysiaSelangor, Malaysia; School of Science, Monash University MalaysiaSelangor, Malaysia.
authors.forename: Han Ming
authors.initials: HM
authors.lastname: Gan
authors.affiliation: Institute for Integrative Biology of the Cell, CEA, CNRS, Univ Paris-Sud, Université Paris-Saclay Orsay, France.
authors.forename: Hélène
authors.initials: H
authors.lastname: Barreteau
authors.affiliation: Institute for Integrative Biology of the Cell, CEA, CNRS, Univ Paris-Sud, Université Paris-Saclay Orsay, France.
authors.forename: Didier
authors.initials: D
authors.lastname: Blanot
authors.affiliation: Institute for Integrative Biology of the Cell, CEA, CNRS, Univ Paris-Sud, Université Paris-Saclay Orsay, France.
authors.forename: Dominique
authors.initials: D
authors.lastname: Mengin-Lecreulx
authors.affiliation: Thomas H. Gosnell School of Life Sciences, Rochester Institute of Technology Rochester, NY, USA.
authors.forename: André O
authors.initials: AO
authors.lastname: Hudson
keywords: MurB; MurC; UDP-N-acetylenolpyruvoylglucosamine reductase; UDP-N-acetylmuramate:l-alanine ligase; Verrucomicrobium spinosum; bacterial cell wall; fusion enzyme; peptidoglycan