Maize Viviparous14: Structure Meets Function

The interdependence of structure and function is a basic principle in biology at all levels from ecosystems to organisms to cells and molecules. The function of an enzyme, as determined by in vitro biochemical analysis of reactions, in vivo genetic study of mutants, and in silico sequence analysis, can give important clues to its structure. Likewise, determination of an enzyme's three-dimensional structure provides valuable insights into its reaction mechanism, substrate preference, and biological function in a cell.

The viviparous14 mutant of maize was identified as a transposon insertion line that showed premature seed germination and altered stomatal regulation, suggesting a defect involving abscisic acid (ABA) (Tan et al., 1997). The VP14 enzyme is a thylakoid membrane protein that catalyzes the committed step in ABA synthesis: oxidative cleavage of the C11,C12 double bond in 9-cis-violaxanthin to form an aldehyde by-product plus xanthoxin, the precursor to ABA. This reaction uses a catalytic non-heme iron ion that binds and activates molecular oxygen, which provides one oxygen atom to each of the two reaction products.

Messing et al. (pages 2970–2980) present a 3.2-Å structure of maize VP14 that reveals a seven-blade β-propeller topped by three α-helices (see figure). The β-propeller is conserved between bacteria and eukaryotes (Kloer and Schulz, 2006) and forms a tunnel containing the active site and the catalytic Fe²⁺ ion. This Fe²⁺ is octahedrally coordinated to four His residues, one water molecule, and one molecule of O₂. The O₂ appears to be bound to the Fe²⁺ in an end-on position, supporting a proposed mechanism in which O₂ extracts an electron from Fe²⁺, producing the highly reactive superoxide anion (O₂⁻), which then attacks and cleaves the C11,C12 double bond. A lack of acidic and basic residues near this double bond argues against a mechanism involving proton transfer and supports the above mechanism involving direct attack by O₂.

The structure of maize VP14 as viewed from the side (A) and top (B) shows a seven-blade β-propeller tunnel containing the active site and catalytic iron plus an α-helical cap important for membrane insertion.

VP14 also has an α-helical cap with a hydrophobic surface patch formed by 25 exposed amino acid residues. Thermodynamic calculations show that the ΔG for transfer of this hydrophobic patch into a membrane is negative (spontaneous), suggesting its interaction with the with the fatty acid interior of the lipid bilayer. Insertion of the hydrophobic patch into the membrane would allow direct access to the active site for the violaxanthin substrate, which is enriched in thylakoid membranes. Surrounding the hydrophobic patch is a crown of charged residues that could interact with the polar head groups of membrane phospholipids and anchor VP14 in the thylakoid membrane.

Although VP14 could not be crystallized with its large hydrophobic substrate, energy minimization analysis produced a predicted structure for the enzyme-substrate complex. The predicted structure shows hydrophobic residues in the tunnel interacting with the isoprene chain and methylenecyclohexane groups of the substrate. Three conserved Phe residues appear to hold the substrate so that the C11,C12 bond about to be cleaved is positioned directly over the catalytic iron.

The authors then compare sequences of VP14 and related plant carotenoid cleavage dioxygenases (CCDs) and produce a structural model of maize CCD1, which has a commercially available substrate. Based on the VP14 structure, they mutated selected residues in CCD1 and identified several that are important for substrate binding, specificity, and enzyme activity. For example, mutation of each of the three conserved Phe residues that are predicted to position the substrate over the catalytic iron dramatically decreased CCD1 activity. This demonstrates the validity of using the VP14 structure as a template for the structure of related plant CCDs and underscores the value of structural determination as a clue to function.