Presentation Information
[P04-501]Dissecting Coenzyme A Biosynthesis in Thermus thermophilus: Insights into an Unprecedented Regulatory Mechanism
○Crispin Marino Fernandez Panlaque1, Jocelyn Nataniel1, Hiroya Tomita2,3, Kentaro Miyazaki2, Kohsuke Honda2,3 (1. Graduate School of Engineering, The University of Osaka (Japan), 2. International Center for Biotechnology, The University of Osaka (Japan), 3. Industrial Biotechnology Initiative Division, Institute for Open and Transdisciplinary Research Initiatives, The University of Osaka (Japan))
Keywords:
Coenzyme A,CoA biosynthesis,Thermus thermophilus,Feedback Inhibition,Regulatory mechanisms
Coenzyme A (CoA) and its thioesters play essential roles in various metabolic pathways. Its biosynthesis typically begins with the phosphorylation of the key precursor pantothenate (Pan) by pantothenate kinase (PanK) producing 4'-phosphopantothenate. This intermediate subsequently undergoes a series of reactions catalyzed by 4'-phosphopantothenoylcysteine synthetase/decarboxylase (PPCS/PPCDC), 4'-phosphopantetheine adenylyltransferase (PPAT), and dephospho-CoA kinase (DPCK), ultimately producing CoA. In contrast to animals, plants and most bacteria can synthesize Pan de novo from 2-oxoisovalerate via the enzymes ketopantoate hydroxymethyltransferase (KPHMT), ketopantoate reductase (KPR), and pantothenate synthetase (PS), bringing the CoA biosynthesis pathway to a total of eight steps. In some bacteria, the homologs of classical KPR are absent, and ketol-acid reductoisomerase (KARI), primarily involved in branched-chain amino acid (BCAAs) biosynthesis, is utilized instead. Given its high energetic cost, CoA biosynthesis is tightly controlled through feedback inhibition, typically at the level of PanK. However, PanKs from certain bacteria, such as the Type III PanK from the thermophilic bacterium Thermus thermophilus are generally insensitive to inhibition by CoA or its derivatives, leaving the regulatory mechanisms of CoA biosynthesis in this organism unclear.
In this study, to shed light on this conundrum, genetic and biochemical analyses were employed to examine enzymes involved in de novo Pan synthesis as prospective regulatory targets. To identify functional KPHMT, KARI, and PS, disruption of the annotated kphmt, kari and ps genes was carried out. The resulting strains displayed Pan auxotrophy, confirming their essentiality in supplying this precursor for CoA synthesis in T. thermophilus HB27. Upon supplementation with BCAAs, Δkari displayed a growth defect in the absence of Pan, indicating that the gene is not essential but important for Pan and BCAAs biosynthesis. Next, the recombinant proteins were prepared and their enzymatic activities were analyzed. The proteins catalyzed specific reactions that reflect those in the CoA biosynthetic pathway. Notably, the enzymatic activities of these proteins were unaffected by CoA or acetyl-CoA (AcCoA), suggesting that none of these proteins serves a regulatory role in CoA biosynthesis. With these results, another compelling candidate, Tt-PPAT, was investigated. Similarly, Tt-PPAT was uninhibited by CoA or AcCoA. Collectively, these findings warrant further investigation of the remaining enzymes in the pathway and also raise the possibility of noncanonical control mechanisms in this pathway, including those operating at the transcriptional level.
In this study, to shed light on this conundrum, genetic and biochemical analyses were employed to examine enzymes involved in de novo Pan synthesis as prospective regulatory targets. To identify functional KPHMT, KARI, and PS, disruption of the annotated kphmt, kari and ps genes was carried out. The resulting strains displayed Pan auxotrophy, confirming their essentiality in supplying this precursor for CoA synthesis in T. thermophilus HB27. Upon supplementation with BCAAs, Δkari displayed a growth defect in the absence of Pan, indicating that the gene is not essential but important for Pan and BCAAs biosynthesis. Next, the recombinant proteins were prepared and their enzymatic activities were analyzed. The proteins catalyzed specific reactions that reflect those in the CoA biosynthetic pathway. Notably, the enzymatic activities of these proteins were unaffected by CoA or acetyl-CoA (AcCoA), suggesting that none of these proteins serves a regulatory role in CoA biosynthesis. With these results, another compelling candidate, Tt-PPAT, was investigated. Similarly, Tt-PPAT was uninhibited by CoA or AcCoA. Collectively, these findings warrant further investigation of the remaining enzymes in the pathway and also raise the possibility of noncanonical control mechanisms in this pathway, including those operating at the transcriptional level.
Comment
To browse or post comments, you must log in.Log in