Highlighted article
Westfall C.; Zubieta C.; Herrman J.; Kapp U.; Nanao M.; and Jez J. (2012) Structural Basis for Prereceptor Modulation of Plant Hormones by GH3 Proteins. Science DOI: 10.1126/science.1221863.
Significance of findings
Westfall, Zubieta et al. have identified exactly how the GH3 protein family physically bind and modify hormones. Moreover their discovery sheds light on the evolution of a simple ancestral protein into a diverse family of enzyme. A highly adaptable general structure is conserved, while specific residues are customised in order to receive the correct substrates. This pattern is seen across the GH3 protein family in many plant species.
Relevant background information
Plant hormone activity is regulated by, among other things, conjugation of the hormone molecule to an amino acid. For example, when IAA is conjugated to aspartic acid or glutamic acid, it is targeted for degradation. IAA-Ala or IAA-Leu, on the other hand, converts it to a hydrolysable storage auxin.
An important group of acyl acid amido synthetases, which catalyse the conjugation reaction in a number of plants including Arabidopsis, maize, rice and mosses, are the GH3 family. It is known that these enzymes carry out adenylation and transferase activities in order to conjugate acyl acid groups, which many plant hormones contain, to amino acids. The acyl acid is first adenylated and then bound to an amino acid to produce the hormone-amino acid conjugate.
AtGH3.11 and AtGH3.12 proteins, characterised in detail in this study, are involved in salicylic acid (SA) and jasmonic acid (JA) metabolism respectively. IAA has also been linked to GH3 family proteins. However the full range of hormones GH3 proteins are capable of modifying is not known, and neither is mechanism of action.
Moss, wild rice, maize, and Arabidopsis thaliana
Results
Westfall, Zubieta et al. put AtGH3.11 and AtGH3.12 into E. coli. Their structures were studied by protein crystallography and site-directed mutagenesis was used to identify the functional domains of the enzymes.
The two GH3 proteins are structurally similar to the adenylating-firefly-luciferase (ANL) enzyme superfamily, which was described by Gulick (2009):
- A large N-terminal domain, made of a β-barrel and two β-sheets flanked by α-helixes.
- A smaller C-terminal domain, consisting of a β-stranded β-sheet, bracketed by two α-helices on each side.
- The ATP/AMP binding site, consisting of a phosphate binding loop and a β-turn-β structure which bind the phosphate group and acyl acid, and a tyrosine which stacks with the adenine ring. The β-turn-β structure contains a glutamine that binds Mg2+ and orients ATP in the correct direction.
- An aspartate containing site that binds the nucleotide ribose.
A flexible hinge loop connecting the N- and C-terminal domains and facilitating conformational changes in response to ligand binding is found in both AtGH3.11 and AtGH3.12, and was confirmed as being conserved in all 19 AtGH3 enzymes by sequence analysis. It is not seen in the ANL super-group. The researchers suggest this is an evolutionary advancement which allows transferase activity as well as adenylation.
When ATP is bound, the hinge swings to ‘open’ the active site, allowing the plant hormone and amino acid to bind as well. The adenylation reaction occurs, and the hormone-AMP product ‘closes’ the hinge. The transferase reaction can proceed, producing the hormone-amino acid conjugate.
Changing the amino acids at certain positions modifies the specificity of the enzyme. In AtGH3.12, Ser96 probably stabilizes the transition state, while Ser328 is key for the transferase step. Arg123 and Tyr 120 form hydrogen bonds with SA. Thr161, Leu 217, Phe218 are all essential active site residues in AtGH3.12.
The exact same structure is seen in AtGH3.11, but specific residues are substituted. Water-mediated hydrogen bonds form between JA-Ile and His328, while number of hydrophobic amino acids form a pocket for JA’s pentenyl tail.
Sequence comparisons of other GH3 family enzymes from species as diverse as soybean, maize, rice and moss, all show similar sequence adaptions that allow for diverse substrate specificity. Westfall, Zubieta et al. devised a phylogeny dividing the GH3 family into eight sub-groups based on four conserved motifs. Within these sub groups, diversity is conferred by amino acid substitutions.
Teaching resources
Although not related to the advanced biochemistry of amino acid conjugation, this activity on auxin and mustard seedlings by SAPS is a good way of introducing plant hormones to secondary school students.
