Abstract
3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase (HMGL) is involved in branched-chain amino acid catabolism leading to acetyl-CoA production. Here, using bioinformatics analyses and protein sequence alignments, we found that in Arabidopsis thaliana a single gene encodes two HMGL isoforms differing in size (51 kDa, HMGL51 and 46 kDa, HMGL46). Similar to animal HMGLs, both isoforms comprised a C-terminal type 1 peroxisomal retention motif, and HMGL51 contained a mitochondrial leader peptide. We observed that only a shortened HMGL (35 kDa, HMGL35) is conserved across all kingdoms of life. Most notably, all plant HMGLs also contained a specific N-terminal extension (P100) that is located between the N-terminal mitochondrial targeting sequence TP35 and HMGL35 and is absent in bacteria and other eukaryotes. Interestingly, using HMGL enzyme assays, we found that rather than HMGL46, homodimeric recombinant HMGL35 is the active enzyme catalyzing acetyl-CoA and acetoacetate synthesis when incubated with (S)-HMG-CoA. This suggested that the plant-specific P100 peptide may inactivate HMGL according to specific physiological requirements. Therefore, we investigated whether the P100 peptide in HMGL46 alters its activity, possibly by modifying the HMGL46 structure. We found that induced expression of a cytosolic HMGL35 version in A. thaliana delays germination and leads to rapid wilting and chlorosis in mature plants. Our results suggest that in plants, P100-mediated HMGL inactivation outside of peroxisomes or mitochondria is crucial, protecting against potentially cytotoxic effects of HMGL activity while it transits to these organelles.
Highlights
3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase (HMGL) is involved in branched-chain amino acid catabolism leading to acetyl-CoA production
Our results suggest that in plants, P100-mediated HMGL inactivation outside of peroxisomes or mitochondria is crucial, protecting against potentially cytotoxic effects of HMGL activity while it transits to these organelles
It is not surprising that over the past several years much attention has been focused on the human enzyme [27]. It essentially has a dietary function by participating in the catabolism of branched-chain amino acids (BCAAs) through the HMG-CoA cycle and ketogenesis in liver, which is crucial during starvation periods [3, 4, 28,29,30,31]
Summary
In P. mevalonii, HMGL participates in a sequence of catabolic reactions that make the use of mevalonic acid (MVA) as a carbon source possible In vertebrates, this enzyme is of paramount importance. It is not surprising that over the past several years much attention has been focused on the human enzyme [27] It essentially has a dietary function by participating in the catabolism of branched-chain amino acids (BCAAs) through the HMG-CoA cycle and ketogenesis in liver, which is crucial during starvation periods [3, 4, 28,29,30,31]. Plant enzymes participate in the biosynthesis of acetyl-CoA via degradation of the BCAA leucine (39 –42), but there is no clear information available that would assign plant HMGLs with metabolic functions to produce ketone bodies. Our study aimed to characterize a plant HMGL and its atypical primary protein structure and provide answers for a raison d’être of this plant distinctiveness
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