The energetic cost of induced fit catalysis: Crystal structures of trypsinogen mutants with enhanced activity and inhibitor affinity

The contribution of induced fit to enzyme specificity has been much debated, although with little experimental data. Here we probe the effect of induced fit on enzyme specificity using the trypsin(ogen) system. BPTI is known to induce trypsinogen to assume a trypsinlike conformation. Correlations are observed between BPTI affinity and the values of k(cat)/K(m) for the hydrolysis of two substrates by eight trypsin(ogen) variants. The slope of both correlations is -1.8. The crystal structures of the BPTI complexes of four variant trypsinogens were also solved. Three of these enzymes, K15A, DeltaI16V17/D194N, and DeltaI16V17/Q156K trypsinogen, are 10- to 100-fold more active than trypsinogen. The fourth variant, DeltaI16V17 trypsinogen, is the lone outlier in the correlations; its activity is lower than expected based on its affinity for BPTI. The S1 site and oxyanion hole, formed by segments 184A-194 and 216-223, are trypsinlike in all of the enzymes. These structural and kinetic data confirm that BPTI induces an active conformation in the trypsin(ogen) variants. Thus, changes in BPTI affinity monitor changes in the energetic cost of inducing a trypsinlike conformation. Although the S1 site and oxyanion hole are similar in all four variants, the N-terminal and autolysis loop (residues 142-152) segments have different interactions for each variant. These results indicate that zymogen activity is controlled by a simple conformational equilibrium between active and inactive conformations, and that the autolysis loop and N-terminal segments control this equilibrium. Together, these data illustrate that induced fit does not generally contribute to enzyme specificity.     

Loss-of-function mutations and inducible RNAi suppression of Arabidopsis LCB2 genes reveal the critical role of sphingolipids in gametophytic and sporophytic cell viability

Serine palmitoyltransferase (SPT) catalyzes the first step in sphingolipid biosynthesis, and downregulation of this enzyme provides a means for exploring sphingolipid function in cells. We have previously demonstrated that Arabidopsis SPT requires LCB1 and LCB2 subunits for activity, as is the case in other eukaryotes. In this study, we show that Arabidopsis has two genes ( AtLCB2a and AtLCB2b ) that encode functional isoforms of the LCB2 subunit. No alterations in sphingolipid content or growth were observed in T-DNA mutants for either gene, but homozygous double mutants were not recoverable, suggesting that these genes are functionally redundant. Reciprocal crosses conducted with Atlcb2a and Atlcb2b mutants indicated that lethality is associated primarily with the inability to transmit the lcb2 null genotype through the haploid pollen. Consistent with this, approximately 50% of the pollen obtained from plants homozygous for a mutation in one gene and heterozygous for a mutation in the second gene arrested during transition from uni-nucleate microspore to bicellular pollen. Ultrastructural analyses revealed that these pollen grains contained aberrant endomembranes and lacked an intine layer. To examine sphingolipid function in sporophytic cells, Arabidopsis lines were generated that allowed inducible RNAi silencing of AtLCB2b in an Atlcb2a mutant background. Studies conducted with these lines demonstrated that sphingolipids are essential throughout plant development, and that lethality resulting from LCB2 silencing in seedlings could be partially rescued by supplying exogenous long-chain bases. Overall, these studies provide insights into the genetic and biochemical properties of SPT and sphingolipid function in Arabidopsis.     

A single amino acid change is responsible for evolution of acyltransferase specificity in bacterial methionine biosynthesis

Bacteria and yeast rely on either homoserine transsuccinylase (HTS, metA) or homoserine transacetylase (HTA; met2) for the biosynthesis of methionine. Although HTS and HTA catalyze similar chemical reactions, these proteins are typically unrelated in both sequence and three-dimensional structure. Here we present the 2.0 A resolution x-ray crystal structure of the Bacillus cereus metA protein in complex with homoserine, which provides the first view of a ligand bound to either HTA or HTS. Surprisingly, functional analysis of the B. cereus metA protein shows that it does not use succinyl-CoA as a substrate. Instead, the protein catalyzes the transacetylation of homoserine using acetyl-CoA. Therefore, the B. cereus metA protein functions as an HTA despite greater than 50% sequence identity with bona fide HTS proteins. This result emphasizes the need for functional confirmation of annotations of enzyme function based on either sequence or structural comparisons. Kinetic analysis of site-directed mutants reveals that the B. cereus metA protein and the E. coli HTS share a common catalytic mechanism. Structural and functional examination of the B. cereus metA protein reveals that a single amino acid in the active site determines acetyl-CoA (Glu-111) versus succinyl-CoA (Gly-111) specificity in the metA-like of acyltransferases. Switching of this residue provides a mechanism for evolving substrate specificity in bacterial methionine biosynthesis. Within this enzyme family, HTS and HTA activity likely arises from divergent evolution in a common structural scaffold with conserved catalytic machinery and homoserine binding sites.     

Production of 22:2<Superscript>Δ5,Δ13</Superscript> and 20:1<Superscript>Δ5</Superscript> in <Emphasis Type="Italic">Brassica carinata</Emphasis> and soybean breeding lines via introduction of <Emphasis Type="Italic">Limnanthes</Emphasis> genes

Seed oils of meadowfoam (Limnanthes douglasii, L. alba) contain very long-chain fatty acids of strategic importance for a number of industrial applications. These include the monoene 20 1⁵ and the diene 22:2^5,13. Engineering of meadowfoam-type oils in other oilseed crops is desirable for the production of these fatty acids as industrial feedstocks. Accordingly, we have targeted Brassica carinata and soybean (Glycine max) to trangenically engineer the biosynthesis of these unusual fatty acids. An L. douglasii seed-specific cDNA (designated Lim Des5) encoding a homolog of acyl-coenzyme A desaturases found in animals, fungi and cyanobacteria was expressed in B. carinata, which resulted in the accumulation of up to 10% 22:2^5,13 in the seed oil. In soybean, co-expression of Lim Des5 with a cDNA (Lim FAE1) encoding an FAEl (elongase complex condensing enzyme) homolog from L. douglasii resulted in the accumulation of 20:1⁵ to approximately 10% of the total fatty acids of seeds. The content of C₂₀ and C₂₂ fatty acids was also increased from <0.5% in non-transformed soybean seeds to >25% in seeds co-expressing the Lim. douglasii Des5 and FAE1 cDNAs. In contrast, expression of the Lim Des5 in Arabidopsis did not produce the expected 20:2^5,11 in the seed oil. Cumulatively, these results demonstrate the utility of soybean and B. carinata for the production of vegetable oils containing novel C₂₀ and C₂₂ fatty acids, and confirm that the preferred substrates of the Lim Des5 are 20:0 and 22:1³, respectively.     

Surface Elevation Dynamics in a Regenerating Mangrove Forest at Homebush Bay,Australia

Following the dieback of an interior portion of a mangrove forest at Homebush Bay, Australia, surface elevation tables and feldspar marker horizons were installed in the impacted, intermediate and control forest to measure vertical accretion, elevation change, and shallow subsidence. The objectives of the study were to determine current vertical accretion and elevation change rates as a guide to understanding mangrove dieback, ascertain the factors controlling surface elevation change, and investigate the sustainability of the mangrove forest under estimated sea-level rise conditions. The study demonstrates that the influences on surface dynamics are more complex than soil accretion and soil autocompaction alone. During strong vegetative regrowth in the impacted forest, surface elevation increase exceeded vertical accretion apparently as a result of belowground biomass production. In addition, surface elevation in all forest zones was correlated with total monthly rainfall during a severe El Niño event, highlighting the importance of rainfall to groundwater recharge and surface elevation. Surface elevation increase for all zones exceeded the 85-year sea level trend for Sydney Harbour. Since mean sea-level also decreased during the El Niño event, the decrease in surface elevation did not translate to an increase in inundation frequency or influence the sustainability of the mangrove forest. These findings indicate that subsurface soil processes such as organic matter accumulation and groundwater flux can significantly influence mangrove surface elevation, and contribute to the long-term sustainability of mangrove systems under a scenario of rising sea levels.     

The plastid clpP gene may not be essential for plant cell viability

The plastid gene clpP is widely regarded as essential for chloroplast function and general plant cell survival. In this note we provide evidence that certain lines of non-photosynthetic maize (Zea mays) Black Mexican Sweet (BMS) suspension cells do not carry clpP in their plastid genomes. We also discuss several incidences in the literature where clpP is either missing or not expressed in other non-green cell lines and plants. We conclude that clpP is not required for general plant cell survival but instead may only be essential for the development and/or function of plastids with active gene expression.     

Fungal responsive fatty acid acetylenases occur widely in evolutionarily distant plant families

The fungal elicitor-induced ELI12 gene from parsley has been previously shown to encode a divergent form of the Delta12-oleic acid desaturase. In this report, we show that the ELI12 gene product is a fatty acid acetylenase or a triple-bond-forming enzyme. Expression of this enzyme in transgenic soybean seeds was accompanied by the accumulation of the Delta12-acetylenic fatty acids, crepenynic and dehydrocrepenynic acids. Using PCR with degenerate oligonucleotides, we also show that homologs of the ELI12 gene are present in other members of the Apiaceae family. In addition, cDNAs for divergent forms of the Delta12-oleic acid desaturase were detected among the expressed sequence tags (ESTs) from English ivy, an Araliaceae species, and sunflower, an Asteraceae species. As with the ELI12 gene, expression of these cDNAs in transgenic soybean embryos was accompanied by the accumulation of crepenynic and dehydrocrepenynic acids. Homologs of the sunflower acetylenase gene were also detected in other Asteraceae species, as revealed by PCR analysis of isolated genomic DNA. Results from Northern blot and EST analyses indicated that the expression of the sunflower gene, like ELI12, was induced by fungal elicitation. Overall, these results demonstrate that expressed genes for Delta12-fatty acid acetylenases occur in at least three plant families, and are responsive to fungal pathogenesis. Natural products derived from crepenynic and dehydrocrepenynic acids that display antifungal, insecticidal, and nematicidal properties are distributed through at least 15 plant families. The acetylenases described here provide probes for chemotaxonomists, and facilitate functional genomic and molecular investigations of these defensive mechanisms.     

Interactions among shrub cover and the soil microclimate may determine future Arctic carbon budgets

Arctic and Boreal terrestrial ecosystems are important components of the climate system because they contain vast amounts of soil carbon (C). Evidence suggests that deciduous shrubs are increasing in abundance, but the implications for ecosystem C budgets remain uncertain. Using midsummer CO₂ flux data from 21 sites spanning 16° of latitude in the Arctic and Boreal biomes, we show that air temperature explains c. one‐half of the variation in ecosystem respiration (ER) and that ER drives the pattern in net ecosystem CO₂ exchange across ecosystems. Woody sites were slightly stronger C sinks compared with herbaceous communities. However, woody sites with warm soils (> 10 °C) were net sources of CO₂, whereas woody sites with cold soils (< 10 °C) were strong sinks. Our results indicate that transition to a shrub‐dominated Arctic will increase the rate of C cycling, and may lead to net C loss if soil temperatures rise.     

Processes Contributing to Resilience of Coastal Wetlands to Sea-Level Rise

The objectives of this study were to identify processes that contribute to resilience of coastal wetlands subject to rising sea levels and to determine whether the relative contribution of these processes varies across different wetland community types. We assessed the resilience of wetlands to sea-level rise along a transitional gradient from tidal freshwater forested wetland (TFFW) to marsh by measuring processes controlling wetland elevation. We found that, over 5 years of measurement, TFFWs were resilient, although some marginally, and oligohaline marshes exhibited robust resilience to sea-level rise. We identified fundamental differences in how resilience is maintained across wetland community types, which have important implications for management activities that aim to restore or conserve resilient systems. We showed that the relative importance of surface and subsurface processes in controlling wetland surface elevation change differed between TFFWs and oligohaline marshes. The marshes had significantly higher rates of surface accretion than the TFFWs, and in the marshes, surface accretion was the primary contributor to elevation change. In contrast, elevation change in TFFWs was more heavily influenced by subsurface processes, such as root zone expansion or compaction, which played an important role in determining resilience of TFFWs to rising sea level. When root zone contributions were removed statistically from comparisons between relative sea-level rise and surface elevation change, sites that previously had elevation rate deficits showed a surplus. Therefore, assessments of wetland resilience that do not include subsurface processes will likely misjudge vulnerability to sea-level rise.