Dataset Information

Bifunctional Malic/Malolactic Enzyme Provides a Novel Mechanism for NADPH-Balancing in Bacillus subtilis.

ABSTRACT: The redox cofactor NADPH is required as a reducing equivalent in about 100 anabolic reactions throughout metabolism. To ensure fitness under all conditions, the demand is fulfilled by a few dehydrogenases in central carbon metabolism that reduce NADP⁺ with electrons derived from the catabolism of nutrients. In the case of Bacillus subtilis growing on glucose, quantitative flux analyses indicate that NADPH production largely exceeds biosynthetic needs, suggesting a hitherto unknown mechanism for NADPH balancing. We investigated the role of the four malic enzymes present in B. subtilis that could bring about a metabolic cycle for transhydrogenation of NADPH into NADH. Using quantitative ¹³C metabolic flux analysis, we found that isoform YtsJ alone contributes to NADPH balancing in vivo and demonstrated relevant NADPH-oxidizing activity by YtsJ in vitro To our surprise, we discovered that depending on NADPH, YtsJ switches activity from a pyruvate-producing malic enzyme to a lactate-generating malolactic enzyme. This switch in activity allows YtsJ to adaptively compensate for cellular NADPH over- and underproduction upon demand. Finally, NADPH-dependent bifunctional activity was also detected in the YtsJ homolog in Escherichia coli MaeB. Overall, our study extends the known redox cofactor balancing mechanisms by providing first-time evidence that the type of catalyzed reaction by an enzyme depends on metabolite abundance.IMPORTANCE A new mechanism for NADPH balancing was discovered in Bacillus subtilis It pivots on the bifunctional enzyme YtsJ, which is known to catalyze NADP-dependent malate decarboxylation. We found that in the presence of excessive NADPH, the same enzyme switches to malolactic activity and creates a transhydrogenation cycle that ultimately converts NADPH to NADH. This provides a regulated mechanism to immediately adjust NADPH/NADP⁺ in response to instantaneous needs.

SUBMITTER: Horl M

PROVIDER: S-EPMC8092299 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Spore-forming bacteria of the orders Bacillales and Clostridiales play a major role in food spoilage and foodborne diseases. When environmental conditions become favorable, these spores can germinate as the germinant receptors located on the spore's inner membrane are activated via germinant binding. This leads to the formation of vegetative cells via germination and subsequent outgrowth and potential deleterious effects on foods. The present report focuses on analysis of the synthesis of the MalS (malic enzyme) protein during Bacillus subtilis spore germination by investigating the dynamics of the presence and fluorescence level of a MalS-GFP (MalS-green fluorescent protein) fusion protein using time-lapse fluorescence microscopy. Our results show an initial increase in MalS-GFP fluorescence intensity within the first 15 min of germination, followed by a discernible drop and stabilization of the fluorescence throughout spore outgrowth as reported previously (L. Sinai, A. Rosenberg, Y. Smith, E. Segev, and S. Ben-Yehuda, Mol Cell 57:695-707, 2015, https://doi.org/10.1016/j.molcel.2014.12.019). However, in contrast to the earlier report, both Western blotting and SILAC (stable isotopic labeling of amino acids in cell culture) analysis showed there was no increase in MalS-GFP levels during the 15 min after the addition of germinants and that MalS synthesis did not begin until more than 90 min after germinant addition. Thus, the increase in MalS-GFP fluorescence early in germination is not due to new protein synthesis but is perhaps due to a change in the physical environment of the spore cores. Our findings also show that different sporulation conditions and spore maturation times affect expression of MalS-GFP and the germination behavior of the spores, albeit to a minor extent, but still result in no changes in MalS-GFP levels early in spore germination.IMPORTANCE The spores formed by Bacillus subtilis remain in a quiescent state for extended periods due to their dormancy and resistance features. Dormancy is linked to a very low level of core water content and a phase-bright state of spores. The present report, focusing on proteins MalS and PdhD (pyruvate dehydrogenase subunit D) and complementary to our companion report published in this issue, aims to shed light on a major dilemma in the field, i.e., whether protein synthesis, in particular that of MalS, takes place in phase-bright spores. Clustered MalS-GFP in dormant spores diffuses throughout the spore as germination proceeds. However, fluorescence intensity measurements, supported by Western blot analysis and SILAC proteomics, confirm that there is no new MalS protein synthesis in bright-phase dormant spores.

Dataset Information

Bifunctional Malic/Malolactic Enzyme Provides a Novel Mechanism for NADPH-Balancing in Bacillus subtilis.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets