Dataset Information

Characterization of a Dimeric Arginase From Zymomonas mobilis ZM4.

ABSTRACT: Many organisms have genes to protect themselves from toxic conditions such as high ethanol and/or ammonia concentrations. When a high ethanol condition is induced to Zymomonas mobilis ZM4, a representative ethanologenic organism, this bacterium overexpresses several genes to overcome this ethanol stress. Among them, we characterized a gene product annotated as an arginase (zmARG) from Z. mobilis ZM4. Even though all of the arginase-determining sequence motifs are not strictly conserved in zmARG, this enzyme converts L-arginine to urea and L-ornithine in the presence of a divalent manganese ion. The revealed high-resolution crystal structure of zmARG shows that it has a typical globular ?/? arginase fold with a protruded C-terminal helix. Two zinc ions reside in the active site, where one metal ion is penta-coordinated and the other has six ligands, discerning this zmARG from the reported arginases with two hexa-liganded metal ions. zmARG forms a dimeric structure in solution as well as in the crystalline state. The dimeric assembly of zmARG is formed mainly by interaction formed between the C-terminal ?-helix of one molecule and the ?/? hydrolase fold of another molecule. The presented findings demonstrate the first reported dimeric arginase formed by the C-terminal tail and has two metal ions coordinated by different number of ligands.

SUBMITTER: Hwangbo SA

PROVIDER: S-EPMC6988801 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Characterization of a Dimeric Arginase From Zymomonas mobilis ZM4.

Hwangbo Seung-A SA Kim Ji-Won JW Jung Sun-Ju SJ Jin Kyeong Sik KS Lee Jie-Oh JO Kim Jeong-Sun JS Park Suk-Youl SY

Frontiers in microbiology 20191126

Many organisms have genes to protect themselves from toxic conditions such as high ethanol and/or ammonia concentrations. When a high ethanol condition is induced to Zymomonas mobilis ZM4, a representative ethanologenic organism, this bacterium overexpresses several genes to overcome this ethanol stress. Among them, we characterized a gene product annotated as an arginase (zmARG) from Z. mobilis ZM4. Even though all of the arginase-determining sequence motifs are not strictly conse ...[more]

PMID: 32038508

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Project description:Zymomonas mobilis is an alpha-proteobacterium that is a promising platform for industrial scale production of biofuels due to its efficient ethanol fermentation and low biomass generation. Z. mobilis is aerotolerant and encodes a complete respiratory electron transport chain, but the benefit of respiration for growth in oxic conditions has never been confirmed, despite decades of research. Growth and ethanol production of wild-type Z. mobilis is poor in oxic conditions indicating that it does not benefit from oxidative phosphorylation. Additionally, in previous studies, aerobic growth improved significantly when respiratory genes were disrupted (ndh) or acquired point mutations (cydA and cydB), even if respiration was significantly reduced by these changes. Here, we obtained clean deletions of respiratory genes ndh and cydAB, individually and in combination, and showed, for the first time, that deletion of cydAB completely inhibited O2 respiration and dramatically reduced growth in oxic conditions. Both respiration and aerobic growth were restored by expressing a heterologous, water-forming NADH oxidase, noxE. Oxygen can have many negative effects, including formation of reactive oxygen species (ROS) or directly inactivating oxygen sensitive enzymes. Our results suggest that the effect of molecular oxygen on enzymes had a greater negative impact on Z. mobilis than formation of ROS. This result shows that the main role of the electron transport chain in Z. mobilis is reducing the intracellular concentration of molecular oxygen, helping to explain why it is beneficial for Z. mobilis to use electron transport chain complexes that have little capacity to contribute to oxidative phosphorylation. IMPORTANCE A key to producing next-generation biofuels is to engineer microbes that efficiently convert non-food materials into drop-in fuels, and to engineer microbes effectively, we must understand their metabolism thoroughly. Zymomonas mobilis is a bacterium that is a promising candidate biofuel producer, but its metabolism remains poorly understood, especially its metabolism when exposed to oxygen. Although Z. mobilis respires with oxygen, its aerobic growth is poor, and disruption of genes related to respiration counterintuitively improves aerobic growth. This unusual result has sparked decades of research and debate regarding the function of respiration in Z. mobilis. Here, we used a new set of mutants to determine that respiration is essential for aerobic growth and likely protects the cells from damage caused by oxygen. We conclude that the respiratory pathway of Z. mobilis should not be deleted from chassis strains for industrial production because this would yield a strain that is intolerant of oxygen, which is more difficult to manage in industrial settings.

Project description:Genome-scale metabolic models have been utilized extensively in the study and engineering of the organisms they describe. Here we present the analysis of a published dataset from pooled transposon mutant fitness experiments as an approach for improving the accuracy and gene-reaction associations of a metabolic model for Zymomonas mobilis ZM4, an industrially relevant ethanologenic organism with extremely high glycolytic flux and low biomass yield. Gene essentiality predictions made by the draft model were compared to data from individual pooled mutant experiments to identify areas of the model requiring deeper validation. Subsequent experiments showed that some of the discrepancies between the model and dataset were caused by polar effects, mis-mapped barcodes, or mutants carrying both wild-type and transposon disrupted gene copies-highlighting potential limitations inherent to data from individual mutants in these high-throughput datasets. Therefore, we analyzed correlations in fitness scores across all 492 experiments in the dataset in the context of functionally related metabolic reaction modules identified within the model via flux coupling analysis. These correlations were used to identify candidate genes for a reaction in histidine biosynthesis lacking an annotated gene and highlight metabolic modules with poorly correlated gene fitness scores. Additional genes for reactions involved in biotin, ubiquinone, and pyridoxine biosynthesis in Z. mobilis were identified and confirmed using mutant complementation experiments. These discovered genes, were incorporated into the final model, iZM4_478, which contains 747 metabolic and transport reactions (of which 612 have gene-protein-reaction associations), 478 genes, and 616 unique metabolites, making it one of the most complete models of Z. mobilis ZM4 to date. The methods of analysis that we applied here with the Z. mobilis transposon mutant dataset, could easily be utilized to improve future genome-scale metabolic reconstructions for organisms where these, or similar, high-throughput datasets are available.

Dataset Information

Characterization of a Dimeric Arginase From Zymomonas mobilis ZM4.

Publications

Characterization of a Dimeric Arginase From <i>Zymomonas mobilis</i> ZM4.

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