Chlorophyte aspartyl aminopeptidases: Ancient origins, expanded families, new locations, and secondary functions.

M18 aspartyl aminopeptidases (DAPs) are well characterized in microbes and animals with likely functions in peptide processing and vesicle trafficking. In contrast, there is a dearth of knowledge on plant aminopeptidases with a preference for proteins and peptides with N-terminal acidic residues. During evolution of the Plantae, there was an expansion and diversification of the M18 DAPs. After divergence of the ancestral green algae from red and glaucophyte algae, a duplication yielded the DAP1 and DAP2 lineages. Subsequently DAP1 genes were lost in chlorophyte algae. A duplication of DAP2-related genes occurred early in green plant evolution. DAP2 genes were retained in land plants and picoeukaryotic algae and lost in green algae. In contrast, DAP2-like genes persisted in picoeukaryotic and green algae, while this lineage was lost in land plants. Consistent with this evolutionary path, Arabidopsis thaliana has two DAP gene lineages (AtDAP1 and AtDAP2). Similar to animal and yeast DAPs, AtDAP1 is localized to the cytosol or vacuole; while AtDAP2 harbors an N-terminal transit peptide and is chloroplast localized. His6-DAP1 and His6-DAP2 expressed in Escherichia coli were enzymatically active and dodecameric with masses exceeding 600 kDa. His6-DAP1 and His6-DAP2 preferentially hydrolyzed Asp-p-nitroanilide and Glu-p-nitroanilide. AtDAPs are highly conserved metallopeptidases activated by MnCl2 and inhibited by ZnCl2 and divalent ion chelators. The protease inhibitor PMSF inhibited and DTT stimulated both His6-DAP1 and His6-DAP2 activities suggesting a role for thiols in the AtDAP catalytic mechanism. The enzymes had distinct pH and temperature optima, as well as distinct kinetic parameters. Both enzymes had high catalytic efficiencies (kcat/Km) exceeding 1.0 x 107 M-1 sec-1. Using established molecular chaperone assays, AtDAP1 and AtDAP2 prevented thermal denaturation. AtDAP1 also prevented protein aggregation and promoted protein refolding. Collectively, these data indicate that plant DAPs have a complex evolutionary history and have evolved new biochemical features that may enable their role in vivo.

Structural insights into chaperone-activity enhancement by a K354E mutation in tomato acidic leucine aminopeptidase.

Tomato plants express acidic leucine aminopeptidase (LAP-A) in response to various environmental stressors. LAP-A not only functions as a peptidase for diverse peptide substrates, but also displays chaperone activity. A K354E mutation has been shown to abolish the peptidase activity but to enhance the chaperone activity of LAP-A. To better understand this moonlighting function of LAP-A, the crystal structure of the K354E mutant was determined at 2.15 Šresolution. The structure reveals that the K354E mutation destabilizes an active-site loop and causes significant rearrangement of active-site residues, leading to loss of the catalytic metal-ion coordination required for the peptidase activity. Although the mutant was crystallized in the same hexameric form as wild-type LAP-A, gel-filtration chromatography revealed an apparent shift from the hexamer to lower-order oligomers for the K354E mutant, showing a mixture of monomers to trimers in solution. In addition, surface-probing assays indicated that the K354E mutant has more accessible hydrophobic areas than wild-type LAP-A. Consistently, computational thermodynamic estimations of the interfaces between LAP-A monomers suggest that increased exposure of hydrophobic surfaces occurs upon hexamer breakdown. These results suggest that the K354E mutation disrupts the active-site loop, which also contributes to the hexameric assembly, and destabilizes the hexamers, resulting in much greater hydrophobic areas accessible for efficient chaperone activity than in the wild-type LAP-A.

Refactoring the Six-Gene Photosystem II Core in the Chloroplast of the Green Algae Chlamydomonas reinhardtii.

Oxygenic photosynthesis provides the energy to produce all food and most of the fuel on this planet. Photosystem II (PSII) is an essential and rate-limiting component of this process. Understanding and modifying PSII function could provide an opportunity for optimizing photosynthetic biomass production, particularly under specific environmental conditions. PSII is a complex multisubunit enzyme with strong interdependence among its components. In this work, we have deleted the six core genes of PSII in the eukaryotic alga Chlamydomonas reinhardtii and refactored them in a single DNA construct. Complementation of the knockout strain with the core PSII synthetic module from three different green algae resulted in reconstitution of photosynthetic activity to 85, 55, and 53% of that of the wild-type, demonstrating that the PSII core can be exchanged between algae species and retain function. The strains, synthetic cassettes, and refactoring strategy developed for this study demonstrate the potential of synthetic biology approaches for tailoring oxygenic photosynthesis and provide a powerful tool for unraveling PSII structure-function relationships.

Chlamydomonas as a model for biofuels and bio-products production.

Developing renewable energy sources is critical to maintaining the economic growth of the planet while protecting the environment. First generation biofuels focused on food crops like corn and sugarcane for ethanol production, and soybean and palm for biodiesel production. Second generation biofuels based on cellulosic ethanol produced from terrestrial plants, has received extensive funding and recently pilot facilities have been commissioned, but to date output of fuels from these sources has fallen well short of what is needed. Recent research and pilot demonstrations have highlighted the potential of algae as one of the most promising sources of sustainable liquid transportation fuels. Algae have also been established as unique biofactories for industrial, therapeutic, and nutraceutical co-products. Chlamydomonas reinhardtii's long established role in the field of basic research in green algae has paved the way for understanding algal metabolism and developing genetic engineering protocols. These tools are now being utilized in C. reinhardtii and in other algal species for the development of strains to maximize biofuels and bio-products yields from the lab to the field.

Structure of tomato wound-induced leucine aminopeptidase sheds light on substrate specificity.

The acidic leucine aminopeptidase (LAP-A) from tomato is induced in response to wounding and insect feeding. Although LAP-A shows in vitro peptidase activity towards peptides and peptide analogs, it is not clear what kind of substrates LAP-A hydrolyzes in vivo. In the current study, the crystal structure of LAP-A was determined to 2.20 Šresolution. Like other LAPs in the M17 peptidase family, LAP-A is a dimer of trimers containing six monomers of bilobal structure. Each monomer contains two metal ions bridged by a water or a hydroxyl ion at the active site. Modeling of different peptides or peptide analogs in the active site of LAP-A reveals a spacious substrate-binding channel that can bind peptides of five or fewer residues with few geometric restrictions. The sequence specificity of the bound peptide is likely to be selected by the structural and chemical restrictions on the amino acid at the P1 and P1' positions because these two amino acids have to bind perfectly at the active site for hydrolysis of the first peptide bond to occur. The hexameric assembly results in the merger of the open ends of the six substrate-binding channels from the LAP-A monomers to form a spacious central cavity allowing the hexameric LAP-A enzyme to simultaneously hydrolyze six peptides containing up to six amino acids each. The hexameric LAP-A enzyme may also hydrolyze long peptides or proteins if only one such substrate is bound to the hexamer because the substrate can extend through the central cavity and the two major solvent channels between the two LAP-A trimers.

Microarray analysis of tomato's early and late wound response reveals new regulatory targets for Leucine aminopeptidase A.

Wounding due to mechanical injury or insect feeding causes a wide array of damage to plant cells including cell disruption, desiccation, metabolite oxidation, and disruption of primary metabolism. In response, plants regulate a variety of genes and metabolic pathways to cope with injury. Tomato (Solanum lycopersicum) is a model for wound signaling but few studies have examined the comprehensive gene expression profiles in response to injury. A cross-species microarray approach using the TIGR potato 10-K cDNA array was analyzed for large-scale temporal (early and late) and spatial (locally and systemically) responses to mechanical wounding in tomato leaves. These analyses demonstrated that tomato regulates many primary and secondary metabolic pathways and this regulation is dependent on both timing and location. To determine if LAP-A, a known modulator of wound signaling, influences gene expression beyond the core of late wound-response genes, changes in RNAs from healthy and wounded Leucine aminopeptidase A-silenced (LapA-SI) and wild-type (WT) leaves were examined. While most of the changes in gene expression after wounding in LapA-SI leaves were similar to WT, overall responses were delayed in the LapA-SI leaves. Moreover, two pathogenesis-related 1 (PR-1c and PR-1a2) and two dehydrin (TAS14 and Dhn3) genes were negatively regulated by LAP-A. Collectively, this study has shown that tomato wound responses are complex and that LAP-A's role in modulation of wound responses extends beyond the well described late-wound gene core.

Plant leucine aminopeptidases moonlight as molecular chaperones to alleviate stress-induced damage.

Leucine aminopeptidases (LAPs) are present in animals, plants, and microbes. In plants, there are two classes of LAPs. The neutral LAPs (LAP-N and its orthologs) are constitutively expressed and detected in all plants, whereas the stress-induced acidic LAPs (LAP-A) are expressed only in a subset of the Solanaceae. LAPs have a role in insect defense and act as a regulator of the late branch of wound signaling in Solanum lycopersicum (tomato). Although the mechanism of LAP-A action is unknown, it has been presumed that LAP peptidase activity is essential for regulating wound signaling. Here we show that plant LAPs are bifunctional. Using three assays to monitor protein protection from heat-induced damage, it was shown that the tomato LAP-A and LAP-N and the Arabidopsis thaliana LAP1 and LAP2 are molecular chaperones. Assays using LAP-A catalytic site mutants demonstrated that LAP-A chaperone activity was independent of its peptidase activity. Furthermore, disruption of the LAP-A hexameric structure increased chaperone activity. Together, these data identify a new class of molecular chaperones and a new function for the plant LAPs as well as suggesting new mechanisms for LAP action in the defense of solanaceous plants against stress.