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Purification and characterization of malate:quinone oxidoreductase from thermophilic Bacillus sp. PS3

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Abstract

Several bacteria possess membrane-bound dehydrogenases other than cytosolic dehydrogenases in their respiratory chains. In many cases, the membrane-bound malate:quinone oxidoreductases (MQOs) are essential for growth. However, these MQOs are absent in mammalian mitochondria, and therefore may be a potential drug target for pathogenic bacteria. To characterize the kinetic properties of MQOs, we purified MQO from Bacillus sp. PS3, which is a gram-positive and thermophilic bacterium, and cloned the gene encoding MQO based on the obtained partial N-terminus sequence. Purified MQOs showed a molecular mass of ~90 kDa, which was estimated using gel filtration, and it consists of two subunits with a molecular mass of ~50 kDa. Phylogenetic analysis showed a high similarity to the MQO of the Geobacillus group rather than the Bacillus group. Additionally, the purified enzyme was thermostable and it retained menaquinol reduction activity at high temperatures. Although it is difficult to conduct experiments using menaquinol because of its instability, we were able to measure the oxidase activity of cytochrome bd-type quinol oxidase by using menaquinol-1 by coupling this molecule with the menaquinol reduction reaction using purified MQOs.

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Author notes

  1. Yoshiki Kabashima

    Present address: Department of Chemistry, School of Medicine, Kyorin University, Mitaka, Tokyo, 181-8611, Japan

Authors and Affiliations

  1. Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, Kawazu 680-4, Iizuka, Fukuoka-ken, 820-8502, Japan

    Yoshiki Kabashima, Nobuhito Sone, Tomoichirou Kusumoto & Junshi Sakamoto

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  1. Yoshiki Kabashima
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  2. Nobuhito Sone
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  3. Tomoichirou Kusumoto
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  4. Junshi Sakamoto
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Corresponding author

Correspondence to Junshi Sakamoto.

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Fig. S1

Nucleic acid and amino acid sequences of purified MQO from thermophilic Bacillus sp. PS3. The square shows the N-terminal sequences of the 54-kDa band (Fig. 2, lane 7) revealed using Edman degradation (see Materials and method section). The bold letters show the putative FAD-binding site. (DOC 49 kb)

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Kabashima, Y., Sone, N., Kusumoto, T. et al. Purification and characterization of malate:quinone oxidoreductase from thermophilic Bacillus sp. PS3. J Bioenerg Biomembr 45, 131–136 (2013). https://doi.org/10.1007/s10863-012-9485-5

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  • DOI: https://doi.org/10.1007/s10863-012-9485-5

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