Michael S. Deal

Industrial‐strength ecology: trade‐offs and opportunities in algal biofuel production

Microalgae represent one of the most promising groups of candidate organisms for replacing fossil fuels with contemporary primary production as a renewable source of energy. Algae can produce many times more biomass per unit area than terrestrial crop plants, easing the competing demands for land with food crops and native ecosystems. However, several aspects of algal biology present unique challenges to the industrial-scale aquaculture of photosynthetic microorganisms. These include high susceptibility to invading aquatic consumers and weeds, as well as prodigious requirements for nutrients that may compete with the fertiliser demands of other crops. Most research on algal biofuel technologies approaches these problems from a cellular or genetic perspective, attempting either to engineer or select algal strains with particular traits. However, inherent functional trade-offs may limit the capacity of genetic selection or synthetic biology to simultaneously optimise multiple functional traits for biofuel productivity and resilience. We argue that a community engineering approach that manages microalgal diversity, species composition and environmental conditions may lead to more robust and productive biofuel ecosystems. We review evidence for trade-offs, challenges and opportunities in algal biofuel cultivation with a goal of guiding research towards intensifying bioenergy production using established principles of community and ecosystem ecology.

Structure and Allostery of the PKA RIIβ Tetrameric Holoenzyme

Keeping a Kinase in Check Cyclic adenosine monophosphate (cAMP)–dependent protein kinase (PKA) is involved in the regulation of several key metabolic pathways. It exists in mammalian cells as an inactive tetramer composed of a regulatory (R) subunit dimer and two catalytic (C) subunits. cAMP binding causes activation by releasing the C subunits. Insight into PKA regulation has come from structures of R and C subunit heterodimers; however, further understanding requires knowledge of the holoenzyme structure. P. Zhang et al. (p. 712) report a high-resolution structure of the RIIβ2:C2 tetramer. The structure reveals interactions at an interface between the two RC heterodimers and provides insight into the mechanism of allosteric activation upon cAMP binding. Details are revealed as to how cyclic adenosine monophosphate (cAMP) binding causes dissociation and activation of cAMP-dependent protein kinase. In its physiological state, cyclic adenosine monophosphate (cAMP)–dependent protein kinase (PKA) is a tetramer that contains a regulatory (R) subunit dimer and two catalytic (C) subunits. We describe here the 2.3 angstrom structure of full-length tetrameric RIIβ2:C2 holoenzyme. This structure showing a dimer of dimers provides a mechanistic understanding of allosteric activation by cAMP. The heterodimers are anchored together by an interface created by the β4-β5 loop in the RIIβ subunit, which docks onto the carboxyl-terminal tail of the adjacent C subunit, thereby forcing the C subunit into a fully closed conformation in the absence of nucleotide. Diffusion of magnesium adenosine triphosphate (ATP) into these crystals trapped not ATP, but the reaction products, adenosine diphosphate and the phosphorylated RIIβ subunit. This complex has implications for the dissociation-reassociation cycling of PKA. The quaternary structure of the RIIβ tetramer differs appreciably from our model of the RIα tetramer, confirming the small-angle x-ray scattering prediction that the structures of each PKA tetramer are different.

Global Consequences of Activation Loop Phosphorylation on Protein Kinase A

Phosphorylation of the activation loop is one of the most common mechanisms for regulating protein kinase activity. The catalytic subunit of cAMP-dependent protein kinase autophosphorylates Thr197 in the activation loop when expressed in Escherichia coli. Although mutation of Arg194 to Ala prevents autophosphorylation, phosphorylation of Thr197 can still be achieved by a heterologous protein kinase, phosphoinositide-dependent protein kinase (PDK1), in vitro. In this study, we examined the structural and functional consequences of adding a single phosphate to the activation loop of cAMP-dependent protein kinase by comparing the wild type C-subunit to the R194A mutant either in the presence or the absence of activation loop phosphorylation. Phosphorylation of Thr197 decreased the Km by ∼15- and 7-fold for kemptide and ATP, respectively, increased the stability of the enzyme as measured by fluorescence and circular dichroism, and enhanced the binding between the C-subunit and IP20, a protein kinase inhibitor peptide. Additionally, deuterium exchange coupled to mass spectrometry was used to compare the structural dynamics of these proteins. All of the regions of the C-subunit analyzed underwent amide hydrogen exchange at a higher or equal rate in the unphosphorylated enzyme compared with the phosphorylated enzyme. The largest changes occurred at the C terminus of the activation segment in the p + 1 loop/APE regions and the αH-αI loop motifs and leads to the prediction of a coordinated phosphorylation-induced salt bridge between two conserved residues, Glu208 and Arg280.

Contribution of Non-catalytic Core Residues to Activity and Regulation in Protein Kinase A

Protein kinase A holoenzyme is comprised of two catalytic (C) and two regulatory (R) subunits which keep the enzyme in an inhibited state before activation by cyclic-AMP. The C-subunit folds into a conserved bi-lobal core flanked by N- and C-terminal tails. We report here characterization of a C-tail loss-of-function mutant, CF327A, and a related suppressor mutant, CF327A/K285P. Phe-327 is the only residue outside the kinase core that binds to the adenine ring of ATP, whereas Lys-285 is ∼45 Å away and lies in an AGC kinase-specific insert. The two mutations were previously identified from a yeast genetic screen, where the F327A mutation was unable to complement cell growth but mutation of K285P in the same allele rescued cell viability. We show that CF327A exhibits significant reduction in catalytic efficiency, which likely explains the observed loss-of-function phenotype. Interestingly, the additional K285P mutation does not restore kinase activity but reduces the inhibitory interaction of the double mutant with RII subunits. The additional K285P mutation, thus, helps to keep a low but uninhibited PKA activity that is sufficient for cell viability. The crystal structure of CF327A/K285P further reveals that recruitment of Phe-327 to the ATP binding pocket not only contributes to the hydrophobic pocket, as previously thought, but also recruits its flanking C-tail region to the kinase core, thereby concertedly positioning the glycine-rich loop and ATP for phosphoryl transfer. The study exemplifies two different ways for regulating cAMP-dependent protein kinase activity through non-conserved residues and sheds light on the structural and functional diversity of the kinase family.

Localization and quaternary structure of the PKA RIβ holoenzyme

Specificity for signaling by cAMP-dependent protein kinase (PKA) is achieved by both targeting and isoform diversity. The inactive PKA holoenzyme has two catalytic (C) subunits and a regulatory (R) subunit dimer (R2:C2). Although the RIα, RIIα, and RIIβ isoforms are well studied, little is known about RIβ. We show here that RIβ is enriched selectively in mitochondria and hypothesized that its unique biological importance and functional nonredundancy will correlate with its structure. Small-angle X-ray scattering showed that the overall shape of RIβ2:C2 is different from its closest homolog, RIα2:C2. The full-length RIβ2:C2 crystal structure allows us to visualize all the domains of the PKA holoenzyme complex and shows how isoform-specific assembly of holoenzyme complexes can create distinct quaternary structures even though the R1:C1 heterodimers are similar in all isoforms. The creation of discrete isoform-specific PKA holoenzyme signaling “foci” paves the way for exploring further biological roles of PKA RIβ and establishes a paradigm for PKA signaling.

A Conserved Glu–Arg Salt Bridge Connects Coevolved Motifs That Define the Eukaryotic Protein Kinase Fold

Eukaryotic protein kinases (EPKs) feature two coevolved structural segments, the Activation segment, which starts with the Asp-Phe-Gly (DFG) and ends with the Ala-Pro-Glu (APE) motifs, and the helical GHI subdomain that comprises αG-αH-αI helices. Eukaryotic-like kinases have a much shorter Activation segment and lack the GHI subdomain. They thus lack the conserved salt bridge interaction between the APE Glu and an Arg from the GHI subdomain, a hallmark signature of EPKs. Although the conservation of this salt bridge in EPKs is well known and its implication in diseases has been illustrated by polymorphism analysis, its function has not been carefully studied. In this work, we use murine cAMP-dependent protein kinase (protein kinase A) as the model enzyme (Glu208 and Arg280) to examine the role of these two residues. We showed that Ala replacement of either residue caused a 40- to 120-fold decrease in catalytic efficiency of the enzyme due to an increase in K(m)(ATP) and a decrease in k(cat). Crystal structures, as well as solution studies, also demonstrate that this ion pair contributes to the hydrophobic network and stability of the enzyme. We show that mutation of either Glu or Arg to Ala renders both mutant proteins less effective substrates for upstream kinase phosphoinositide-dependent kinase 1. We propose that the Glu208-Arg280 pair serves as a center hub of connectivity between these two structurally conserved elements in EPKs. Mutations of either residue disrupt communication not only between the two segments but also within the rest of the molecule, leading to altered catalytic activity and enzyme regulation.