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Identification and partial characterization of a novel UDP-N-acetylenolpyruvoylglucosamine reductase/UDP-N-acetylmuramate: L-alanine ligase fusion enzyme from Verrucomicrobium spinosum DSM 4136T

Naqvi, Kubra F., Patin, Delphine, Wheatley, Matthew S., Savka, Michael A., Dobson, Renwick C. J., Gan, Han Ming, Barreteau, Helene, Blanot, Didier, Mengin-Lecreulx, Dominique and Hudson, Andre O. 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, Frontiers in microbiology, vol. 7, pp. 1-13, doi: 10.3389/fmicb.2016.00362.

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Title Identification and partial characterization of a novel UDP-N-acetylenolpyruvoylglucosamine reductase/UDP-N-acetylmuramate: L-alanine ligase fusion enzyme from Verrucomicrobium spinosum DSM 4136T
Author(s) Naqvi, Kubra F.
Patin, Delphine
Wheatley, Matthew S.
Savka, Michael A.
Dobson, Renwick C. J.
Gan, Han Ming
Barreteau, Helene
Blanot, Didier
Mengin-Lecreulx, Dominique
Hudson, Andre O.
Journal name Frontiers in microbiology
Volume number 7
Article ID 362
Start page 1
End page 13
Total pages 13
Publisher Frontiers Research Foundation
Place of publication Lausanne, Switzerland
Publication date 2016-03-23
ISSN 1664-302X
Keyword(s) MurB
MurC
UDP-N-acetylenolpyruvoylglucosamine reductase
UDP-N-acetylmuramate:L-alanine ligase
fusion enzyme
bacterial cell wall
peptidoglycan
Verrucomicrobium spinosum
Summary 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.
Language eng
DOI 10.3389/fmicb.2016.00362
Field of Research 060503 Microbial Genetics
060501 Bacteriology
HERDC Research category C1.1 Refereed article in a scholarly journal
ERA Research output type C Journal article
Copyright notice ©2016, The Authors
Free to Read? Yes
Use Rights Creative Commons Attribution licence
Persistent URL http://hdl.handle.net/10536/DRO/DU:30101935

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Every reasonable effort has been made to ensure that permission has been obtained for items included in DRO. If you believe that your rights have been infringed by this repository, please contact drosupport@deakin.edu.au.