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. 2020 Aug 21;15(8):2281-2288.
doi: 10.1021/acschembio.0c00507. Epub 2020 Aug 12.

Cyanobacterial Dihydroxyacid Dehydratases Are a Promising Growth Inhibition Target

Affiliations

Cyanobacterial Dihydroxyacid Dehydratases Are a Promising Growth Inhibition Target

Peilan Zhang et al. ACS Chem Biol. .

Abstract

Microbes are essential to the global ecosystem, but undesirable microbial growth causes issues ranging from food spoilage and infectious diseases to harmful cyanobacterial blooms. The use of chemicals to control microbial growth has achieved significant success, while specific roles for a majority of essential genes in growth control remain unexplored. Here, we show the growth inhibition of cyanobacterial species by targeting an essential enzyme for the biosynthesis of branched-chain amino acids. Specifically, we report the biochemical, genetic, and structural characterization of dihydroxyacid dehydratase from the model cyanobacterium Synechocystis sp. PCC 6803 (SnDHAD). Our studies suggest that SnDHAD is an oxygen-stable enzyme containing a [2Fe-2S] cluster. Furthermore, we demonstrate that SnDHAD is selectively inhibited in vitro and in vivo by the natural product aspterric acid, which also inhibits the growth of representative bloom-forming Microcystis and Anabaena strains but has minimal effects on microbial pathogens with [4Fe-4S] containing DHADs. This study suggests DHADs as a promising target for the precise growth control of microbes and highlights the exploration of other untargeted essential genes for microbial management.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1.
Figure 1.
The biosynthetic pathway to branched-chain amino acids. The reaction of IlvD (DHAD shown in red) is depicted in the dashed box. LeuA, isopropylmalate synthase; LeuB, isopropylmalate dehydrogenase; LeuC/D, isopropylmalate isomerase.
Figure 2.
Figure 2.
Biochemical characterization of SnDHAD. A: Recombinant EcDHAD and SnDHAD showed expected molecular weights in SDS-PAGE analysis and (B:) different oligomeric states in native gel analysis. C: Relative catalytic activities of SnDHAD and its mutants. The amount of KIV in the wild type (WT) enzyme reaction was set as 100% for normalizing the relative activities of others. The blank had no enzyme. D: Relative catalytic activities of SnDHAD and EcDHAD after exposed to air at room temperature (RT) and 4 °C. Their residual activities at indicated days were determined. E: Metal dependence of the SnDHAD reaction. The enzyme activity in the reaction without any divalent metal was set as 1 for normalizing its relative activity when supplemented with serial metal ions and EDTA.
Figure 3.
Figure 3.
In vivo functional characterization of DHADs. A: Growth inhibition of TA and AA toward Synechocystis. BCAAs (B) at 0.05 mM were supplemented to BG-11 medium. B-C: Growth inhibition of TA (50 mM) and AA (10 μM) toward wild type (WT) E. coli, ΔilvD and ΔilvD::SnDHAD mutants in M9 medium. BCAAs at 1 mM were supplemented. Cell optical density at 600 nm was measured at indicated time points.
Figure 4.
Figure 4.
The crystal structure of SnDHAD. A: Ribbon diagram of the monomer with the two subdomains highlights. B: Ribbon representation of the SnDHAD monomer with modeled active site and substrate DHIV. Expanded view of the active site of SnDHAD showing modeled DHIV coordinated to the [2Fe-2S] cluster and Mg2+.
Figure 5.
Figure 5.
DHADs are a promising target for selective growth control. A: The growth of Synechocystis, Microcystis aeruginosa NIES 298 and Anabaena sp. PCC 7120 was inhibited by AA in a dose-dependent manner. The optical density (730 nm) of each cyanobacterium in BG-11 was determined and set as 100% for normalizing the strain growth in the presence of AA and/or 0.05 mM BCAAs. B: Microbial pathogens showed varied responses to AA (0.1 mM). The optical density (600 nm) of each strain in minimal medium was determined and set as 100% for normalizing the strain growth in the presence of AA and/or 1 mM BCAAs.

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