Branched-chain Α-keto Dehydrogenase Research Articles

Utilization of multiple substrates by butyrate kinase from Listeria monocytogenes

Listeria monocytogenes, the causative agent of listeriosis, can build up to dangerous levels in refrigerated foods potentially leading to expensive product recalls. An important aspect of the bacterium's growth at low temperatures is its ability to increase the branched-chain fatty acid anteiso C15:0 content of its membrane at lower growth temperatures, which imparts greater membrane fluidity. Mutants in the branched-chain α-keto dehydrogenase (bkd) complex are deficient in branched-chain fatty acids (BCFAs,) but these can be restored by feeding C4 and C5 branched-chain carboxylic acids (BCCAs). This suggests the presence of an alternate pathway for production of acyl CoA precursors for fatty acid biosynthesis. We hypothesize that the alternate pathway is composed of butyrate kinase (buk) and phosphotransbutyrylase (ptb) encoded in the bkd complex which produce acyl CoA products by their sequential action through the metabolism of carboxylic acids. We determined the steady state kinetics of recombinant His-tagged Buk using 11 different straight-chain and BCCA substrates in the acyl phosphate forming direction. Buk demonstrated highest catalytic efficiency with pentanoate as the substrate. Low product formation observed with acetate (C2) and hexanoate (C6) as the substrates indicates that Buk is not involved in either acetate metabolism or long chain carboxylic acid activation. We were also able to show that Buk catalysis occurs through a ternary complex intermediate. Additionally, Buk demonstrates a strong preference for BCCAs at low temperatures. These results indicate that Buk may be involved in the activation and assimilation of exogenous carboxylic acids for membrane fatty acid biosynthesis.

Tissue-specific and Nutrient Regulation of the Branched-chain α-Keto Acid Dehydrogenase Phosphatase, Protein Phosphatase 2Cm (PP2Cm)

Branched-chain amino acid (BCAA) homeostasis is maintained through highly regulated catabolic activities where the rate-limiting step is catalyzed by branched-chain α-keto dehydrogenase (BCKD). Our previous study has identified a mitochondria-targeted protein phosphatase, PP2Cm, as the BCKD phosphatase and thus serves as a key regulator for BCAA catabolism. In this report, we performed comprehensive molecular and biochemical studies of PP2Cm regulation using both in vivo and in vitro systems. We show that PP2Cm expression is highly enriched in brain, heart, liver, kidney, and diaphragm, but low in skeletal muscle. The PP2Cm expression is regulated at the transcriptional level in response to nutrient status. Furthermore, we have established that PP2Cm interacts with the BCKD E2 subunit and competes with the BCKD kinase in a substrate-dependent and mutually exclusive manner. These data suggest that BCAA homeostasis is at least in part contributed by nutrient-dependent PP2Cm expression and interaction with the BCKD complex. Finally, a number of human PP2Cm single nucleotide polymorphic changes as identified in the public data base can produce either inactive or constitutive active mutant phosphatases, suggesting that putative PP2Cm mutations may contribute to BCAA catabolic defects in human.