Reductionism in the study of enzyme adaptation.

One of the principal goals of comparative biology is the elucidation of mechanisms by which organisms adapt to different environments. The study of enzyme structure, function, and stability has contributed significantly to this effort, by revealing adaptation at a molecular level. Comparative biochemistry, including enzymology, necessarily pursues a reductionist approach in describing the function and structure of biomolecules, allowing more straightforward study of molecular systems by removing much of the complexity of their biological milieu. Although this reductionism has allowed a remarkable series of discoveries linking chemical processes to metabolism and to whole-organism function in the context of the environment, it also has the potential to mislead when careful consideration is not made of the simplifying assumptions inherent to such research. In this review, a brief history of the growth of enzymology, its reliance on a reductionist philosophy, and its contributions to our understanding of biological systems is given. Examples then are provided of research techniques, based on a reductionist approach, that have advanced our knowledge about enzyme adaptation to environmental stresses, including stability assays, enzyme kinetics, and the impact of solute composition on enzyme function. In each case, the benefits of the reductionist nature of the approach is emphasized, notable advances are described, but potential drawbacks due to inherent oversimplification of the study system are also identified.

Thermal stability and structure of glyceraldehyde-3-phosphate dehydrogenase from the coral Acropora millepora.

Corals are vulnerable to increasing ocean temperatures. It is known that elevated temperatures lead to the breakdown of an essential mutualistic relationship with photosynthetic algae. The molecular mechanisms of this temperature-dependent loss of symbiosis are less well understood. Here, the thermal stability of a critical metabolic enzyme, glyceraldehyde-3-phosphate dehydrogenase, from the stony coral Acropora millepora was found to increase significantly in the presence of its cofactor NAD+. Determination of the structure of the cofactor-enzyme complex (PDB ID 6PX2) revealed variable NAD+ occupancy across the four monomers of the tetrameric enzyme. The structure of the fully occupied monomers was compared to those with partial cofactor occupancy, identifying regions of difference that may account for the increased thermal stability.

A year in the salt marsh: Seasonal changes in gill protein expression in the temperate intertidal mussel Geukensia demissa.

Organisms living in temperate and polar regions experience extensive seasonal changes in the physical and biotic environment, including temperature, insolation, and food availability, among other factors. Sessile intertidal organisms respond to such seasonal fluctuations largely through physiological and biochemical means, because their behavioral responses are severely limited. In this study, we used a proteomic approach to examine changes in seasonal protein expression of gill from the intertidal mussel Geukensia demissa, a keystone species of the western Atlantic salt marsh, over the course of one year. Gill tissue of mussels collected in summer had the greatest number of proteins significantly increased in abundance (37 of 592 spots detected on two-dimensional polyacrylamide gels), although autumn mussels revealed a comparable proportion of up-regulated proteins (31 spots). In contrast, the number of proteins changing in abundance in winter and spring mussels were substantially smaller (15 and 9, respectively). Identification of these proteins revealed both expected and unanticipated changes to the proteome. Maintenance of gill cilia dominates in the summer when filter-feeding is most active, as evidenced by cytoskeletal proteins such as tektin-4 and tubulin isoforms; a signal of protection from heat stress is also present in summer (e.g., heat shock cognate 70). In autumn oxidative stress protection (peroxiredoxin-5 and manganese-containing superoxide dismutase) and aerobic ATP synthetic capacity (ATP synthase subunits a and delta) appear to increase. In winter a signal of cold-induced oxidative stress is apparent (Mn-SOD and NADP-dependent isocitrate dehydrogenase), perhaps in association with heavy metal toxicity and exposure to pathogens. Gill tissue from spring shows relatively little environmental acclimatization, other than a possible increase in protein synthesis capacity.

Rapid proteomic responses to a near-lethal heat stress in the salt marsh mussel Geukensia demissa.

Acute heat stress perturbs cellular function on a variety of levels, leading to protein dysfunction and aggregation, oxidative stress and loss of metabolic homeostasis. If these challenges are not overcome quickly, the stressed organism can die. To better understand the earliest tissue-level responses to heat stress, we examined the proteomic response of gill from Geukensia demissa, an extremely eurythermal mussel from the temperate intertidal zone of eastern North America. We exposed 15°C-acclimated individuals to an acute near-lethal heat stress (45°C) for 1 h, and collected gill samples from 0 to 24 h of recovery. The changes in protein expression we found reveal a coordinated physiological response to acute heat stress: proteins associated with apoptotic processes were increased in abundance during the stress itself (i.e. at 0 h of recovery), while protein chaperones and foldases increased in abundance soon after (3 h). The greatest number of proteins changed abundance at 6 h; these included oxidative stress proteins and enzymes of energy metabolism. Proteins associated with the cytoskeleton and extracellular matrix also changed in abundance starting at 6 h, providing evidence of cell proliferation, migration and tissue remodeling. By 12 h, the response to acute heat stress was diminishing, with fewer stress and structural proteins changing in abundance. Finally, the proteins with altered abundances identified at 24 h suggest a return to the pre-stress anabolic state.

Adaptations of protein structure and function to temperature: there is more than one way to 'skin a cat'.

Sensitivity to temperature helps determine the success of organisms in all habitats, and is caused by the susceptibility of biochemical processes, including enzyme function, to temperature change. A series of studies using two structurally and catalytically related enzymes, A4-lactate dehydrogenase (A4-LDH) and cytosolic malate dehydrogenase (cMDH) have been especially valuable in determining the functional attributes of enzymes most sensitive to temperature, and identifying amino acid substitutions that lead to changes in those attributes. The results of these efforts indicate that ligand binding affinity and catalytic rate are key targets during temperature adaptation: ligand affinity decreases during cold adaptation to allow more rapid catalysis. Structural changes causing these functional shifts often comprise only a single amino acid substitution in an enzyme subunit containing approximately 330 residues; they occur on the surface of the protein in or near regions of the enzyme that move during catalysis, but not in the active site; and they decrease stability in cold-adapted orthologs by altering intra-molecular hydrogen bonding patterns or interactions with the solvent. Despite these structure-function insights, we currently are unable to predict a priori how a particular substitution alters enzyme function in relation to temperature. A predictive ability of this nature might allow a proteome-wide survey of adaptation to temperature and reveal what fraction of the proteome may need to adapt to temperature changes of the order predicted by global warming models. Approaches employing algorithms that calculate changes in protein stability in response to a mutation have the potential to help predict temperature adaptation in enzymes; however, using examples of temperature-adaptive mutations in A4-LDH and cMDH, we find that the algorithms we tested currently lack the sensitivity to detect the small changes in flexibility that are central to enzyme adaptation to temperature.

High-pressure-low-temperature cryostat designed for use with fourier transform infrared spectrometers and time-resolved infrared spectroscopy.

The design for a new high-pressure-low-temperature infrared (IR) cell for performing experiments using conventional Fourier transform infrared or fast laser-based time-resolved infrared spectroscopy, in a range of solvents, is described. The design builds upon a commercially available compressor and cold end (Polycold PCC(®) and CryoTiger(®)), which enables almost vibration-free operation, ideal for use with sensitive instrumentation. The design of our cell and cryostat allows for the study of systems at temperatures from 77 to 310 K and at pressures up to 250 bar. The CaF2 windows pass light from the mid-IR to the ultraviolet (UV), enabling a number of experiments to be performed, such as Raman, UV-visible absorption spectroscopy, and time-resolved techniques where sample excitation/probing using continuous wave or pulsed lasers is required. We demonstrate the capabilities of this cell by detailing two different applications: (i) the reactivity of a range of Group V-VII organometallic alkane complexes using time-resolved spectroscopy on the millisecond timescale and (ii) the gas-to-liquid phase transition of CO2 at low temperature, which is applicable to measurements associated with transportation issues related to carbon capture and storage.

Changes in protein expression in the salt marsh mussel Geukensia demissa: evidence for a shift from anaerobic to aerobic metabolism during prolonged aerial exposure.

During aerial exposure (emersion), most sessile intertidal invertebrates experience cellular stress caused by hypoxia, and the amount and types of hypoxia-induced stress will differ as exposure time increases, likely leading to altered metabolic responses. We examined proteomic responses to increasing emersion times and decreasing recovery (immersion) times in the mussel Geukensia demissa, which occurs in salt marshes along the east coast of North America. Individuals are found above mean tide level, and can be emersed for over 18 h during spring tides. We acclimated mussels to full immersion at 15°C for 4 weeks, and compared changes in gill protein expression between groups of mussels that were continually immersed (control), were emersed for 6 h and immersed during recovery for 18 h (6E/18R), were emersed for 12 h and recovered for 12 h (12E/12R), or were emersed for 18 h with a 6 h recovery (18E/6R). We found clear differences in protein expression patterns among the treatments. Proteins associated with anaerobic fermentation increased in abundance in 6E/18R but not in 12E/12R or 18E/6R. Increases in oxidative stress proteins were most apparent in 12E/12R, and in 18E/6R changes in cytoskeletal protein expression predominated. We conclude that G. demissa alters its strategy for coping with emersion stress over time, relying on anaerobic metabolism for short- to medium-duration exposure, but switching to an air-gaping strategy for long-term exposure, which reduces hypoxia stress but may cause structural damage to gill tissue.

Latitudinal variation in protein expression after heat stress in the salt marsh mussel Geukensia demissa.

Individuals of a broadly distributed species often experience significantly different environmental conditions depending on location. For example, the mussel Geukensia demissa occurs intertidally from the Gulf of St. Lawrence to central Florida; within this range, northern populations are exposed to temperatures cold enough to freeze the tissue, whereas southern populations can experience temperatures approaching the species' upper lethal limit. Thus, G. demissa provides an ideal system with which to study physiological variation in conspecifics occurring across a broad latitudinal range. We collected G. demissa at five sites from Maine to Florida, encompassing a range of 1900 km, and have used a proteomic approach to describe how protein expression varies in individuals from the different locations. We acclimated individuals from each site to common conditions (18°C) for 4 weeks, and exposed a subset of these to acute heat stress (40°C). We separated gill proteins using two-dimensional gel electrophoresis and quantified abundances of the resulting protein spots. Among mussels acclimated to 18°C protein, expression profiles were more similar among individuals from the same site than among sites, but there was no discernible correlation with latitude. In contrast, after acute heat stress, protein expression among mussels from different locations varied substantially, with 31 of 448 proteins changing in abundance in the northernmost (Maine) group, compared with 5-11 proteins in the four southern groups. Identification of 27 of these proteins revealed five functional clusters: chaperones, cytoskeletal proteins, oxidative stress proteins, regulatory proteins, and a translation initiation factor. Across these functional categories, the two northernmost groups, Maine and New York, showed the greatest number of proteins that changed significantly in abundance, as well as the greatest fold-change in abundance for many of the proteins. We conclude that the northern populations of G. demissa are physiologically distinct from the southern groups, and that the differences in protein-expression profiles are consistent with greater sensitivity to heat stress to the north.

Proteomic responses of blue mussel (Mytilus) congeners to temperature acclimation.

The ability to acclimate to variable environmental conditions affects the biogeographic range of species, their success at colonizing new habitats, and their likelihood of surviving rapid anthropogenic climate change. Here we compared responses to temperature acclimation (4 weeks at 7, 13 and 19°C) in gill tissue of the warm-adapted intertidal blue mussel Mytilus galloprovincialis, an invasive species in the northeastern Pacific, and the cold-adapted M. trossulus, the native congener in the region, to better understand the physiological differences underlying the ongoing competition. Using two-dimensional gel electrophoresis and tandem mass spectrometry, we showed that warm acclimation caused changes in cytoskeletal composition and proteins of energy metabolism in both species, consistent with increasing rates of filtration and respiration due to increased ciliary activity. During cold acclimation, changes in cytoskeletal proteins were accompanied by increasing abundances of oxidative stress proteins and molecular chaperones, possibly because of the increased production of aldehydes as indicated by the upregulation of aldehyde dehydrogenase. The cold-adapted M. trossulus showed increased abundances of molecular chaperones at 19°C, but M. galloprovincialis did not, suggesting that the two species differ in their long-term upper thermal limits. In contrast, the warm-adapted M. galloprovincialis showed a stronger response to cold acclimation than M. trossulus, including changes in abundance in more proteins and differing protein expression profiles between 7 and 13°C, a pattern absent in M. trossulus. In general, increasing levels of oxidative stress proteins inversely correlate with modifications in Krebs cycle and electron transport chain proteins, indicating a trade-off between oxidative stress resistance and energy production. Overall, our results help explain why M. galloprovincialis has replaced M. trossulus in southern California over the last century, but also suggest that M. trossulus may maintain a competitive advantage at colder temperatures. Anthropogenic global warming may reinforce the advantage M. galloprovincialis has over M. trossulus in the warmer parts of the latter's historical range.

Coping with thermal challenges: physiological adaptations to environmental temperatures.

Temperature profoundly influences physiological responses in animals, primarily due to the effects on biochemical reaction rates. Since physiological responses are often exemplified by their rate dependency (e.g., rate of blood flow, rate of metabolism, rate of heat production, and rate of ion pumping), the study of temperature adaptations has a long history in comparative and evolutionary physiology. Animals may either defend a fairly constant temperature by recruiting biochemical mechanisms of heat production and utilizing physiological responses geared toward modifying heat loss and heat gain from the environment, or utilize biochemical modifications to allow for physiological adjustments to temperature. Biochemical adaptations to temperature involve alterations in protein structure that compromise the effects of increased temperatures on improving catalytic enzyme function with the detrimental influences of higher temperature on protein stability. Temperature has acted to shape the responses of animal proteins in manners that generally preserve turnover rates at animals' normal, or optimal, body temperatures. Physiological responses to cold and warmth differ depending on whether animals maintain elevated body temperatures (endothermic) or exhibit minimal internal heat production (ectothermic). In both cases, however, these mechanisms involve regulated neural and hormonal over heat flow to the body or heat flow within the body. Examples of biochemical responses to temperature in endotherms involve metabolic uncoupling mechanisms that decrease metabolic efficiency with the outcome of producing heat, whereas ectothermic adaptations to temperature are best exemplified by the numerous mechanisms that allow for the tolerance or avoidance of ice crystal formation at temperatures below 0°C.

Function of muscle-type lactate dehydrogenase and citrate synthase of the Galápagos marine iguana, Amblyrhynchus cristatus, in relation to temperature.

The Galápagos marine iguana, Amblyrhynchus cristatus, is unique among lizards in foraging subtidally, leading to activity across a broad range of ambient temperatures ( approximately 14-40 degrees C). To determine whether the marine iguana shows any biochemical changes consistent with maintaining enzyme function at both warm and cold body temperatures, we examined the function of the aerobic enzyme citrate synthase (CS) and the muscle isoform of the anaerobic enzyme lactate dehydrogenase (A(4)-LDH) in A. cristatus and a confamilial species, Iguana iguana, from 14 to 46 degrees C. We also deduced amino acid sequences from cDNA of each enzyme. In CS, despite two amino acid substitutions, we found no difference in the apparent Michaelis-Menten constant K(m) of oxaloacetate at any temperature, indicating that the substrate affinity of CS in A. cristatus has not adapted to changes in thermal environment. In A(4)-LDH, we used site-directed mutagenesis to show that the substitutions T9A and I283V (A. cristatus --> I. iguana) individually have no effect on kinetics, but together significantly decrease the K(m) of pyruvate and catalytic rate constant (k(cat)) of the A. cristatus ortholog. Thus, our data show that A. cristatus A(4)-LDH has not become cold adapted in response to this species' aquatic foraging behavior, and instead may be consistent with moderate warm adaptation with respect to the I. iguana ortholog.

Sublethal exposure to UV radiation affects respiration rates of the freshwater cladoceran Daphnia catawba.

We examined the effects of UV radiation (UVR) on metabolic rates of the freshwater cladoceran Daphnia catawba. We exposed D. catawba to UVB for 12 h in a lamp phototron at levels of 2.08 and 4.16 kJ m(-2) both with and without concomitant exposure to UVA and visible photorepair radiation (PRR). We also included a group that received PRR only and a dark control group. Respiration rates were measured for 6 h following exposure. Respiration rates increased by 31.8% relative to the dark control at the lowest level of UVB stress (2.08 kJ m(-2) UVB with PRR), whereas respiration was inhibited by 70.3% at the highest stress level (4.16 kJ m(-2) UVB without PRR). Survival rates in the group that received PRR only and the group exposed to 2.08 kJ m(-2) and PRR were not significantly different from that in the control group; however, the survival rate was reduced for all other UVR exposures. We hypothesize that enhanced respiration rates reflect energetic costs related to repair of cellular components damaged by sublethal levels of UVR. Increases in respiration rate of the magnitude we found in our experiment could significantly reduce energetic reserves available for growth and reproduction, especially in cases where these costs are incurred repeatedly during a series of days with high levels of UVR.

Temperature sensitivities of cytosolic malate dehydrogenases from native and invasive species of marine mussels (genus Mytilus): sequence-function linkages and correlations with biogeographic distribution.

The blue mussel Mytilus galloprovincialis, a native of the Mediterranean Sea, has invaded the west coast of North America in the past century, displacing the native blue mussel, Mytilus trossulus, from most of its former habitats in central and southern California. The invasive success of M. galloprovincialis is conjectured to be due, in part, to physiological adaptations that enable it to outperform M. trossulus at high temperatures. We have examined the structure and function of the enzyme cytosolic malate dehydrogenase (cMDH) from these species, as well as from the more distantly related ribbed mussel, Mytilus californianus, to characterize the effects of temperature on kinetic properties thought to exhibit thermal adaptation. The M. trossulus cMDH ortholog differs from the other cMDHs in a direction consistent with cold adaptation, as evidenced by a higher and more temperature-sensitive Michaelis-Menten constant for the cofactor NADH (Km(NADH)). This difference results from minor changes in sequence: the M. trossulus ortholog differs from the M. galloprovincialis ortholog by only two substitutions in the 334 amino acid monomer, and the M. californianus and M. trossulus orthologs differ by five substitutions. In each case, only one of these substitutions is non-conservative. To test the effects of individual substitutions on kinetic properties, we used site-directed mutagenesis to create recombinant cMDHs. Recombinant wild-type M. trossulus cMDH (rWT) has high Km(NADH) compared with mutants incorporating the non-conservative substitutions found in M. californianus and M. galloprovincialis - V114H and V114N, respectively - demonstrating that these mutations are responsible for the differences found in substrate affinity. Turnover number (kcat) is also higher in rWT compared with the two mutants, consistent with cold adaptation in the M. trossulus ortholog. Conversely, rWT and V114H appear more thermostable than V114N. Based on a comparison of Km(NADH) and kcat values among the orthologs, we propose that immersion temperatures are of greater selective importance in adapting kinetic properties than the more extreme temperatures that occur during emersion. The relative warm adaptation of M. galloprovincialis cMDH may be one of a suite of physiological characters that enhance the competitive ability of this invasive species in warm habitats.

Decreases in activation energy and substrate affinity in cold-adapted A4-lactate dehydrogenase: evidence from the Antarctic notothenioid fish Chaenocephalus aceratus.

Enzyme function is strongly affected by temperature, and orthologs from species adapted to different thermal environments often show temperature compensation in kinetic properties. Antarctic notothenioid fishes live in a habitat of constant, extreme cold (-1.86 +/- 2 degrees C), and orthologs of the enzyme A4-lactate dehydrogenase (A4-LDH) in these species have adapted to this environment through higher catalytic rates, lower Arrhenius activation energies (Ea), and increases in the apparent Michaelis constant for the substrate pyruvate (Km(PYR)). Here, site-directed mutagenesis was used to determine which amino acid substitutions found in A4-LDH of the notothenioid Chaenocephalus aceratus, with respect to orthologs from warm-adapted teleosts, are responsible for these adaptive changes in enzyme function. Km(PYR) was measured in eight single and two double mutants, and Ea was tested in five single and two double mutants in the temperature range 0 degrees C-20 degrees C. Of the four mutants that had an effect on these parameters, two increased Ea but did not affect Km(PYR) (Gly224Ser, Ala310Pro), and two increased both Ea and Km(PYR) (Glu233Met, Gln317Val). The double mutants Glu233Met/Ala310Pro and Glu233Met/Gln317Val increased Km(PYR) and Ea to levels not significantly different from the A4-LDH of a warm temperate fish (Gillichthys mirabilis, habitat temperature 10 degrees C-35 degrees C). The four single mutants are associated with two alpha-helices that move during the catalytic cycle; those that affect Ea but not Km(PYR) are further from the active site than those that affect both parameters. These results provide evidence that (1) cold adaptation in A4-LDH involves changes in mobility of catalytically important molecular structures; (2) these changes may alter activation energy alone or activation energy and substrate affinity together; and (3) the extent to which these parameters are affected may depend on the location of the substitutions within the mobile alpha-helices, perhaps due to differences in proximity to the active site.

Temperature adaptation in Gillichthys (Teleost: Gobiidae) A(4)-lactate dehydrogenases: identical primary structures produce subtly different conformations.

Alternative conformations of proteins underlie a variety of biological phenomena, from prion proteins that cause spongiform encephalopathies to membrane channel proteins whose conformational changes admit or exclude specific ions. In this paper, we argue that conformational differences within globular 'housekeeping' enzymes may allow rapid adaptation to novel environments. Muscle-type lactate dehydrogenases (A(4)-LDHs) from the gobies Gillichthys seta and G. mirabilis have identical amino acid sequences but show potentially adaptive differences in substrate affinity (apparent Michaelis constants for pyruvate, K(m)(PYR)) as well as differences in thermal stability. We examined the A(4)-LDH of each species using fluorescence spectroscopy, near- and far-ultraviolet circular dichroism (CD) spectroscopy and hydrogen/deuterium exchange (H/D) Fourier-transform infrared spectroscopy to determine whether structural differences were apparent, the extent to which structural differences could be related to differences in conformational flexibility and whether specific changes in secondary or tertiary structure could be defined. The fluorescence spectra and far-ultraviolet CD spectra of the A(4)-LDH from the two species were indistinguishable, suggesting that the two conformations are very similar in secondary and tertiary structure. Apparent melting temperatures (T(m)) followed by fluorescence and CD spectroscopy confirmed that the G. mirabilis A(4)-LDH is more thermally stable than the G. seta form. H/D exchange kinetics of Gillichthys A(4)-LDH was described using double-exponential regression; at 20 degrees C, G. seta A(4)-LDH has a higher exchange constant, indicating a more flexible and open structure. At 40 degrees C, the difference in H/D exchange constants disappears. Second-derivative analysis of H/D exchange infrared spectra indicates that alpha-helical, but not beta-sheet structure, differs in conformational flexibility between the two forms. Second-derivative ultraviolet spectra indicate that at least one of the five tyrosyl residues in the Gillichthys LDH-A monomer is located in a more hydrophobic environment in the G. mirabilis form. Homology models of A(4)-LDH indicate that Tyr246 is the most likely candidate to experience a modified environment because it is involved in subunit contacts within the homotetramer and sits in a hinge between a static alpha-helix and one involved in catalytic conformational changes. Subtle differences in conformation around this residue probably play a role both in altered flexibility and in the potentially adaptive differences in kinetics between the two A(4)-LDH forms.

Phylogenetic relationships and biochemical properties of the duplicated cytosolic and mitochondrial isoforms of malate dehydrogenase from a teleost fish, Sphyraena idiastes.

Unlike birds and mammals, teleost fish express two paralogous isoforms (paralogues) of cytosolic malate dehydrogenase (cMDH; EC 1.1.1.37; NAD+: malate oxidoreductase) whose evolutionary relationships to the single cMDH of tetrapods are unknown. We sequenced complementary DNAs for both cMDHs and the mitochondrial isoform (mMDH) of the fish Sphyraena idiastes (south temperate barracuda) and compared the sequences, kinetic properties, and thermal stabilities of the three isoforms with those of mammalian orthologues. Both fish cMDHs comprise 333 residues and have subunit masses of approximately 36 kDa. One cytosolic isoform, cMDH-S, was significantly more heat-stable than either the other cMDH (cMDH-L) or mMDH. In contradiction to the generally accepted model of vertebrate cMDH evolution, our phylogenetic analysis indicates that the duplication of the fish cytosolic paralogues occurred after the divergence of the lineages leading to teleosts and tetrapods. cMDH-L and cMDH-S differed in optimal concentrations of substrates and cofactors and apparent Michaelis-Menten constants, suggesting that the two paralogues may play distinct physiological roles. Differences in intrinsic thermal stability among MDH paralogues may reflect different degrees of stabilization in vivo by extrinsic stabilizers, notably protein concentration in the case of mMDH. Thermal stabilities of porcine mMDH and cMDH-L, but not cMDH-S, were significantly increased when denaturation was measured at a high protein (bovine serum albumin; BSA) concentration, but the BSA-induced stabilization reduced the catalytic activity.