Interrogating the role of the two distinct fructose-bisphosphate aldolases of Bacillus methanolicus by site-directed mutagenesis of key amino acids and gene repression by CRISPRi

Schultenkämper K, Gütle DD, Gil Lopez M, Keller LB, Zhang L, Einsle O, Jacquot J-P, Wendisch VF (2021)
Front Microbiol 12: 669220.

Zeitschriftenaufsatz | Veröffentlicht | Englisch
 
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Schultenkämper, KerstinUniBi; Gütle, Desirée D. ; Gil Lopez, MarinaUniBi; Keller, Laura B.; Zhang, Lin; Einsle, Oliver; Jacquot, Jean-Pierre; Wendisch, Volker F.UniBi
Abstract / Bemerkung
The Gram-positive Bacillus methanolicus shows plasmid-dependent methylotrophy. This facultative ribulose monophosphate (RuMP) cycle methylotroph possesses two fructose bisphosphate aldolases (FBA) with distinct kinetic properties. The chromosomally encoded FBAC is the major glycolytic aldolase. The gene for the major gluconeogenic aldolase FBAP is found on the natural plasmid pBM19 and is induced during methylotrophic growth. The crystal structures of both enzymes were solved at 2.2 Å and 2.0 Å, respectively, and they suggested amino acid residue 51 to be crucial for binding fructose-1,6-bisphosphate (FBP) as substrate and amino acid residue 140 for active site zinc atom coordination. As FBAC and FBAP differed at these positions, site-directed mutagenesis (SDM) was performed to exchange one or both amino acid residues of the respective proteins. The aldol cleavage reaction was negatively affected by the amino acid exchanges that led to a complete loss of glycolytic activity of FBAP. However, both FBAC and FBAP maintained gluconeogenic aldol condensation activity, and the amino acid exchanges improved the catalytic efficiency of the major glycolytic aldolase FBAC in gluconeogenic direction at least 3-fold. These results confirmed the importance of the structural differences between FBAC and FBAP concerning their distinct enzymatic properties. In order to investigate the physiological roles of both aldolases, the expression of their genes was repressed individually by CRISPR interference (CRISPRi). The fbaC RNA levels were reduced by CRISPRi, but concomitantly the fbaP RNA levels were increased. Vice versa, a similar compensatory increase of the fbaC RNA levels was observed when fbaP was repressed by CRISPRi. In addition, targeting fbaP decreased tktP RNA levels since both genes are cotranscribed in a bicistronic operon. However, reduced tktP RNA levels were not compensated for by increased RNA levels of the chromosomal transketolase gene tktC.
Stichworte
transketolase; methylotrophy; glycolysis; gluconeogenesis; CRISPR interference; fructose-1; 6-bisphosphate aldolase; sedoheptulose 1; 7-bisphosphate aldolase
Erscheinungsjahr
2021
Zeitschriftentitel
Front Microbiol
Band
12
Art.-Nr.
669220
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Open-Access-Publikationskosten wurden durch die Universität Bielefeld gefördert.
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https://pub.uni-bielefeld.de/record/2953457

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Schultenkämper K, Gütle DD, Gil Lopez M, et al. Interrogating the role of the two distinct fructose-bisphosphate aldolases of Bacillus methanolicus by site-directed mutagenesis of key amino acids and gene repression by CRISPRi. Front Microbiol. 2021;12: 669220.
Schultenkämper, K., Gütle, D. D., Gil Lopez, M., Keller, L. B., Zhang, L., Einsle, O., Jacquot, J. - P., et al. (2021). Interrogating the role of the two distinct fructose-bisphosphate aldolases of Bacillus methanolicus by site-directed mutagenesis of key amino acids and gene repression by CRISPRi. Front Microbiol, 12, 669220. https://doi.org/10.3389/fmicb.2021.669220
Schultenkämper, Kerstin, Gütle, Desirée D., Gil Lopez, Marina, Keller, Laura B., Zhang, Lin, Einsle, Oliver, Jacquot, Jean-Pierre, and Wendisch, Volker F. 2021. “Interrogating the role of the two distinct fructose-bisphosphate aldolases of Bacillus methanolicus by site-directed mutagenesis of key amino acids and gene repression by CRISPRi”. Front Microbiol 12: 669220.
Schultenkämper, K., Gütle, D. D., Gil Lopez, M., Keller, L. B., Zhang, L., Einsle, O., Jacquot, J. - P., and Wendisch, V. F. (2021). Interrogating the role of the two distinct fructose-bisphosphate aldolases of Bacillus methanolicus by site-directed mutagenesis of key amino acids and gene repression by CRISPRi. Front Microbiol 12:669220.
Schultenkämper, K., et al., 2021. Interrogating the role of the two distinct fructose-bisphosphate aldolases of Bacillus methanolicus by site-directed mutagenesis of key amino acids and gene repression by CRISPRi. Front Microbiol, 12: 669220.
K. Schultenkämper, et al., “Interrogating the role of the two distinct fructose-bisphosphate aldolases of Bacillus methanolicus by site-directed mutagenesis of key amino acids and gene repression by CRISPRi”, Front Microbiol, vol. 12, 2021, : 669220.
Schultenkämper, K., Gütle, D.D., Gil Lopez, M., Keller, L.B., Zhang, L., Einsle, O., Jacquot, J.-P., Wendisch, V.F.: Interrogating the role of the two distinct fructose-bisphosphate aldolases of Bacillus methanolicus by site-directed mutagenesis of key amino acids and gene repression by CRISPRi. Front Microbiol. 12, : 669220 (2021).
Schultenkämper, Kerstin, Gütle, Desirée D., Gil Lopez, Marina, Keller, Laura B., Zhang, Lin, Einsle, Oliver, Jacquot, Jean-Pierre, and Wendisch, Volker F. “Interrogating the role of the two distinct fructose-bisphosphate aldolases of Bacillus methanolicus by site-directed mutagenesis of key amino acids and gene repression by CRISPRi”. Front Microbiol 12 (2021): 669220.
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