3-Methylcrotonyl-coenzyme A carboxylase is a component of the mitochondrial leucine catabolic pathway in plants

Abstract

3-Methylcrotonyl-coenzyme A carboxylase (MCCase) is a mitochondrial biotin-containing enzyme whose metabolic function is not well understood in plants. In soybean (Glycine max) seedlings the organ-specific and developmentally induced changes in MCCase expression are regulated by mechanisms that control the accumulation of MCCase mRNA and the activity of the enzyme. During soybean cotyledon development, when seed-storage proteins are degraded, leucine (Leu) accumulation peaks transiently at 8 d after planting. The coincidence between peak MCCase expression and the decline in Leu content provides correlative evidence that MCCase is involved in the mitochondrial catabolism of Leu. Direct evidence for this conclusion was obtained from radiotracer metabolic studies using extracts from isolated mitochondria. These experiments traced the metabolic fate of [U-14C]Leu and NaH14CO3, the latter of which was incorporated into methylglutaconyl-coenzyme A (CoA) via MCCase. These studies directly demonstrate that plant mitochondria can catabolize Leu via the following scheme: Leu --> alpha-ketoisocaproate --> isovaleryl-CoA --> 3-methylcrotonyl-CoA --> 3-methylglutaconyl-CoA --> 3-hydroxy-3-methylglutaryl-CoA --> acetoacetate + acetyl-CoA. These findings demonstrate for the first time, to our knowledge, that the enzymes responsible for Leu catabolism are present in plant mitochondria. We conclude that a primary metabolic role of MCCase in plants is the catabolism of Leu.

Figure 1

Potential metabolic functions of MCCase. MCCase catalyzes the ATP-dependent carboxylation of MC-CoA to form MG-CoA. This reaction may be required in the catabolism of Leu to acetoacetate and acetyl-CoA (reactions 1–6). MCCase may also function to convert mevalonate (MVA) to acetoacetate and acetyl-CoA (via isopentenyl pyrophosphate [IPP] and 3-methylcrotonoic acid) by the “mevalonate shunt.” A third function of MCCase may be as part of an isoprenoid catabolic pathway (via geranoyl-CoA). Reactions 4 to 6 are common to all three processes. The products of these processes, acetoacetate and acetyl-CoA, can be further metabolized to isoprenoids, polyketide derivatives (e.g. flavonoids, stilbenoids), and fatty acids, to Glc in tissues engaging the glyoxylate cycle, or respired to CO₂ in the tricarboxylic acid cycle. The enzymes of Leu catabolism are: (a) branched-chain amino acid aminotransferase, (b) BCKDH complex, (c) branched-chain acyl-CoA dehydrogenase, (d) MCCase, (e) MG-CoA hydratase, and (f) HMG-CoA lyase. The asterisks denote the carbon atom expected to be radioactively labeled when NaH¹⁴CO₃ is supplied for the MCCase reaction.

Figure 2

MCCase activity and the accumulation of the biotin-containing subunit of MCCase in soybean seedlings at 13 DAP. A, Organs of a soybean seedling. B, MCCase activity (mean of five experiments ± se). C, Western blot probed with antiserum to detect the biotin-containing subunit of MCCase. D, Western blot identical to that shown in C, but instead probed with ¹²⁵I-streptavidin to detect the biotin prosthetic group on the biotin-containing subunit of MCCase. In C and D protein samples were loaded on the basis of equal MCCase activity (0.1 nmol/min) to detect differences in the catalytic efficiency of the enzyme among organs. The data presented in C and D were gathered from a single experiment; five replicates of this experiment gave similar results.

Figure 3

Effect of cotyledon development on MCCase activity and Leu, protein, and chlorophyll accumulation. A, Chlorophyll and total protein contents. B, MCCase activity and free Leu content. C, Western blot probed with antiserum to detect the biotin-containing subunit of MCCase. D, Western blot identical to that shown in C, but instead probed with ¹²⁵I-streptavidin to detect the biotin prosthetic group on the biotin-containing subunit of MCCase. In C and D protein samples were loaded on the basis of equal MCCase activity (0.1 nmol/min) to detect differences in the catalytic efficiency of the enzyme during cotyledon development. The data presented in A and B are means ± se from three replicates. The data presented in C and D were gathered from a single experiment; three replicates of this experiment gave similar results.

Figure 5

Kinetics of the incorporation of radioactively labeled bicarbonate into acid-stable metabolites by mitochondrial extracts in the presence of MC-CoA and cofactors. Time course of the incorporation of radioactivity into acid-stable products (A), MG-CoA (B), HMG-CoA (C), or acetoacetate (D). Incubations were carried out either without or with 10 μg of avidin (to inhibit MCCase activity), which was added 2.5 min after the start of the reaction (arrows). Each time point is an independent sample and represents two replicates. Because of the rapid incorporation of NaH¹⁴CO₃, the background radioactivity was impossible to obtain. Therefore, the 0-min time point was derived from samples incubated for 1 min in the presence of 10 μg of avidin.

Figure 4

Steady-state levels of the mRNA coding for the biotin-containing subunit of MCCase in organs of soybean seedlings at 13 DAP and during cotyledon development. Ethidium bromide-stained agarose gel containing approximately equal amounts (20 μg) of RNA, isolated from various organs (A) or from cotyledons at different stages of development (B). Northern blot depicting the accumulation of the mRNA coding for the biotin-containing subunit of MCCase in various organs (C) or in cotyledons at different stages of development (D). The data presented in this figure were gathered from a single experiment. Five replicates (A and C) and three replicates (B and D) of these experiments gave similar results.

Figure 6

Separation of radioactive metabolites by HPLC. Representative chromatograms depicting metabolites arising from the catabolism of MC-CoA by mitochondrial extracts in the presence of NaH¹⁴CO₃ and cofactors after 5-min (A) or 45-min (B) incubations. After 5 min [¹⁴C]MG-CoA and [¹⁴C]HMG-CoA were at similar levels and [¹⁴C]acetoacetate was barely detectable. After 45 min [¹⁴C]HMG-CoA had exceeded [¹⁴C]MG-CoA and [¹⁴C]acetoacetate had increased. Representative chromatograms depicting metabolites arising from the catabolism of [U-¹⁴C]Leu by mitochondrial extracts in the presence of 10 μg of avidin after a 2-h (C) or 6-h (D) incubation. After 2 h α-[¹⁴C]-KIC accumulated to a substantial level and [¹⁴C]IV-CoA accumulation was in progress. After 6 h the level of α-[¹⁴C]-KIC diminished, indicating that the rate of its disappearance had exceeded its formation. [¹⁴C]IV-CoA continued to accumulate and [¹⁴C]MC-CoA appeared. [¹⁴C]Isovaleric acid was likely a decarboxylation product of α-[¹⁴C]-KIC, possibly arising nonenzymatically.