Saori Kosono Graduate School of Agricultural and Life Science; Collaborative Research Institute for Innovative Microbiology, The University of Tokyo
Research objective
Pyruvate dehydrogenase (PDH) and 2-oxoglutarate dehydrogenase (ODH) are critical enzymes in central carbon metabolism. In Corynebacterium glutamicum , which is utilized for industrial production of amino acids, including L-glutamate, an unusual hybrid complex consisting of CgE1p (thiamine diphosphate-dependent pyruvate dehydrogenase, AceE), CgE2 (dihydrolipoamide acetyltransferase, AceF), CgE3 (dihydrolipoamide dehydrogenase, Lpd) along with CgE1o (thiamine diphosphate-dependent 2-oxoglutarate dehydrogenase, OdhA) has been suggested. In this study, the molecular architecture and function of the PDH-ODH hybrid complex from C. glutamicum was evaluated to elucidate a novel mechanism for a balanced control of PDH and ODH activities. The potential impact of lysine acetylation on the regulation of the PDH-ODH hybrid complex was also studied.
Methods
In vitro reconstitution of PDH and ODH was performed using recombinant proteins CgE1p, CgE1o, CgE2, and CgE3, which were produced in E. coli. The CgE2-CgE3 subcomplex was copurified using N-terminal His-tagged CgE3. Gel filtration chromatography and pull-down assays were used to determine the assembly and subunit interactions of the complex. Ultracentrifugation analysis was performed using C. glutamicum lysates to detect PDH and ODH complexes in vivo. K391 variant strains of C. glutamicum were constructed using a two- step homologous recombination strategy.
Results
1. In vitro reconstitution and characterization of the PDH-ODH hybrid complex 1)
PDH and ODH activities were successfully reconstituted in vitro using recombinant E. coli proteins CgE1p, CgE1o, CgE2, and CgE3. CgE2 formed a stable complex with CgE3 (CgE2– CgE3 subcomplex) in vitro, which was copurified using N-terminal His-tagged CgE3. The molecular mass of the CgE2–CgE3 subcomplex was estimated to be 780 kDa by gel filtration chromatography with molecular size standards, which suggested six copies of CgE2 and eight copies of CgE3, giving two CgE2 trimers and four CgE3 dimers (Figure 1). Six copies of CgE2 in the core are rather compact compared to E. coli PDH and ODH that have twenty-four copies of E2. The mixture sample containing CgE1o and CgE2–CgE3 provided a new peak fraction with a higher molecular mass than the individual CgE1o and CgE2–CgE3 and ODH activity. This indicated a reconstituted ODH complex, the molecular mass of which was estimated to be approximately 1 MDa. Meanwhile, the mixture sample containing CgE1p and CgE2–CgE3 exhibited PDH activity, but it was lost after gel filtration chromatography. This indicated that the association of CgE1p with CgE2–CgE3 was too weak to be retained during gel filtration, and thus CgPDH was a fragile complex. Given that CgE1p and CgE1o subunits are associated with CgE2–CgE3 in a complex, CgE1p (or CgE1o) may compete with CgE1o (or CgE1p) for binding to CgE2–CgE3, resulting in competitive inhibition of ODH and PDH via CgE1p and CgE1o, respectively. As expected, CgE1p- and CgE1o-dependent inhibition of ODH and PDH activities, respectively, were observed. The inhibitory effect of CgE1o on PDH activity was stronger than that of CgE1p on ODH, which was consistent with the results obtained by gel filtration chromatography analysis. These results suggest that CgE1p and CgE1o associate with CgE2-CgE3 with different affinities, and that the change in the ratio of CgE1p and CgE1o associated with the complex may be involved in the balanced control of PDH and ODH activities. It was previously considered that a CgE1o dimer associates with CgE2–CgE3, as reported for other ODHs; however, it was found that CgE1o existed mainly as a hexamer in solution. CgE1p existed as a dimer in solution, but it migrated to higher molecular mass fractions independent of CgE2 in ultracentrifugation analyses using lysates, suggesting that CgE1p may interact with unknown protein(s) other than CgE2.
2. Characterization of a lysine acetylation site in the peripheral subunit-binding domain
(PSBD) of the CgE2 subunit²)
Protein lysine acetylation is emerging as a mechanism that controls the activity of metabolic enzymes. Lys391 in the PSBD of CgE2 was found to be acetylated by our acetylome analysis. Interestingly, the CgE2–K391R variant strain showed increased L-glutamate production compared to the wild-type and acetylation-mimic K391Q variant strains. This result suggests that acetylation of Lys391 affects L-glutamate production. To understand the reason for the increased L-glutamate production in the K391R strain, the effect of K391 substitutions on the assembly and activity of PDH and ODH was analyzed. The acetylation-mimic K391Q substitution, but not the K391R, severely reduced the interaction of CgE2 with CgE1p and CgE3, but did not affect the interaction with CgE1o. This indicated that Lys391 is a critical residue in the assembly of CgE1p and CgE3 into the complex, suggesting that its acetylation affects the assembly of PDH and ODH (Figure 1). Both K391R and K391Q substitutions also affected enzymatic activities, especially PDH. Kinetic analyses of PDH and ODH using C. glutamicum lysates showed that the apparent V max value of PDH decreased with the K391R variant and further decreased with the K391Q variant. Interestingly, the apparent K m values for pyruvate and 2-oxoglutarate decreased in both K391R and K391Q variants. The calculated PDH activity of the wild-type, K391R, and K391Q variants toward 1 mM pyruvate were estimated using the obtained apparent kinetic parameters and the Michaelis–Menten equation. The activity was higher in K391R than in wild-type and K391Q. Pyruvate production was lower in K391R than in wild-type and K391Q. Collectively, these results suggest that the K391R variant may efficiently convert pyruvate to acetyl-CoA to increase the carbon flux into the citric acid cycle, which may be the reason for the increased glutamate production in the K391R strain.
Conclusion
This study revealed several unique features of PDH and ODH hybrid complexes from C . glutamicum . Since the hybrid complex is compact and composed of a smaller number of subunits compared to E. coli PDH and ODH, the change in the subunit stoichiometry may easily affect the balance between PDH and ODH activities, which critically influences the central carbon flux. It has also been suggested that lysine acetylation has an impact on PDH and ODH function and L-glutamate production.