Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 286, Issue 33, Pages 28794-28801Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M111.262519
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Funding
- National Science Foundation [MCB-0845162, MCB-0904215]
- Dept. of Energy Office of Biological and Environmental Research [DE-AC02-06CH11357]
- American Society of Plant Biologists
- National Institutes of Health-MARC uSTAR
- Div Of Molecular and Cellular Bioscience
- Direct For Biological Sciences [0904215] Funding Source: National Science Foundation
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The methionine chain-elongation pathway is required for aliphatic glucosinolate biosynthesis in plants and evolved from leucine biosynthesis. In Arabidopsis thaliana, three 3-isopropylmalate dehydrogenases (AtIPMDHs) play key roles in methionine chain-elongation for the synthesis of aliphatic glucosinolates (e. g. AtIPMDH1) and leucine (e. g. AtIPMDH2 and AtIPMDH3). Here we elucidate the molecular basis underlying the metabolic specialization of these enzymes. The 2.25 angstrom resolution crystal structure of AtIPMDH2 was solved to provide the first detailed molecular architecture of a plant IPMDH. Modeling of 3-isopropylmalate binding in the AtIPMDH2 active site and sequence comparisons of prokaryotic and eukaryotic IPMDH suggest that substitution of one active site residue may lead to altered substrate specificity and metabolic function. Site-directed mutagenesis of Phe-137 to a leucine in AtIPMDH1 (AtIPMDH1-F137L) reduced activity toward 3-(2'-methylthio)ethylmalate by 200-fold, but enhanced catalytic efficiency with 3-isopropylmalate to levels observed with AtIPMDH2 and AtIPMDH3. Conversely, the AtIPMDH2-L134F and AtIPMDH3-L133F mutants enhanced catalytic efficiency with 3-(2'-methylthio) ethylmalate similar to 100-fold and reduced activity for 3-isopropylmalate. Furthermore, the altered in vivo glucosinolate profile of an Arabidopsis ipmdh1 T-DNA knock-out mutant could be restored to wild-type levels by constructs expressing AtIPMDH1, AtIPMDH2-L134F, or AtIPMDH3-L133F, but not by AtIPMDH1-F137L. These results indicate that a single amino acid substitution results in functional divergence of IPMDH in planta to affect substrate specificity and contributes to the evolution of specialized glucosinolate biosynthesis from the ancestral leucine pathway.
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