Georgina Garza-Ramos

Exploring the evolutionary route of the acquisition of betaine aldehyde dehydrogenase activity by plant ALDH10 enzymes: implications for the synthesis of the osmoprotectant glycine betaine

BackgroundPlant ALDH10 enzymes are aminoaldehyde dehydrogenases (AMADHs) that oxidize different ω-amino or trimethylammonium aldehydes, but only some of them have betaine aldehyde dehydrogenase (BADH) activity and produce the osmoprotectant glycine betaine (GB). The latter enzymes possess alanine or cysteine at position 441 (numbering of the spinach enzyme, SoBADH), while those ALDH10s that cannot oxidize betaine aldehyde (BAL) have isoleucine at this position. Only the plants that contain A441- or C441-type ALDH10 isoenzymes accumulate GB in response to osmotic stress. In this work we explored the evolutionary history of the acquisition of BAL specificity by plant ALDH10s.ResultsWe performed extensive phylogenetic analyses and constructed and characterized, kinetically and structurally, four SoBADH variants that simulate the parsimonious intermediates in the evolutionary pathway from I441-type to A441- or C441-type enzymes. All mutants had a correct folding, average thermal stabilities and similar activity with aminopropionaldehyde, but whereas A441S and A441T exhibited significant activity with BAL, A441V and A441F did not. The kinetics of the mutants were consistent with their predicted structural features obtained by modeling, and confirmed the importance of position 441 for BAL specificity. The acquisition of BADH activity could have happened through any of these intermediates without detriment of the original function or protein stability. Phylogenetic studies showed that this event occurred independently several times during angiosperms evolution when an ALDH10 gene duplicate changed the critical Ile residue for Ala or Cys in two consecutive single mutations. ALDH10 isoenzymes frequently group in two clades within a plant family: one includes peroxisomal I441-type, the other peroxisomal and non-peroxisomal I441-, A441- or C441-type. Interestingly, high GB-accumulators plants have non-peroxisomal A441- or C441-type isoenzymes, while low-GB accumulators have the peroxisomal C441-type, suggesting some limitations in the peroxisomal GB synthesis.ConclusionOur findings shed light on the evolution of the synthesis of GB in plants, a metabolic trait of most ecological and physiological relevance for their tolerance to drought, hypersaline soils and cold. Together, our results are consistent with smooth evolutionary pathways for the acquisition of the BADH function from ancestral I441-type AMADHs, thus explaining the relatively high occurrence of this event.

The folding pathway of glycosomal triosephosphate isomerase: Structural insights into equilibrium intermediates

The guanidine hydrochloride‐induced conformational transitions of glycosomal triosephosphate isomerase (TIM) were monitored with functional, spectroscopic, and hydrodynamic measurements. The equilibrium folding pathway was found to include two intermediates (N2↔I2↔2M↔2U). According to this model, the conformational stability parameters of TIM are as follows: ΔGI2‐N2 = 5.5 ± 0.6, ΔG2M‐I2=19.6 ± 1.6, and ΔG2U‐2M = 14.7 ± 3.1 kcal mol−1. The I2 state is compact (αSR = 0.8); it is able to bind 8‐anilinonaphthalene‐1‐sulfonic acid ANS and it is composed of ∼45% of α‐helix and tertiary structure content compared with the native enzyme; however, it is unable to bind the transition‐state analog 2‐phosphoglycolate. Conversely, the 2M state lacks detectable tertiary contacts, possesses ∼10% of the native α‐helical content, is significantly expanded (αSR = 0.2), and has low affinity for ANS. We studied the effect of mutating cysteine residues on the structure and stability of I2 and 2M. Three mutants were made: C39A, C126A, and C39A/C126A. The replacement of C39, which is located at β2, was found to be neutral. The I2–C126A state, however, was prone to aggregation and exhibited an emission maximum that was 3‐nm red‐shifted compared with the I2–wild type, indicating solvent exposure of W90 at β4. Our results suggest that the I2 state comprises the (βα)1‐4β5 module in which the conserved C126 residue located at β5 defines the boundary of the folded segment. We propose a folding pathway that highlights the remarkable thermodynamic stability of this glycosomal enzyme. Proteins 2012;

Moonlighting Peptides with Emerging Function

Hunter-killer peptides combine two activities in a single polypeptide that work in an independent fashion like many other multi-functional, multi-domain proteins. We hypothesize that emergent functions may result from the combination of two or more activities in a single protein domain and that could be a mechanism selected in nature to form moonlighting proteins. We designed moonlighting peptides using the two mechanisms proposed to be involved in the evolution of such molecules (i.e., to mutate non-functional residues and the use of natively unfolded peptides). We observed that our moonlighting peptides exhibited two activities that together rendered a new function that induces cell death in yeast. Thus, we propose that moonlighting in proteins promotes emergent properties providing a further level of complexity in living organisms so far unappreciated.

Potassium and Ionic Strength Effects on the Conformational and Thermal Stability of Two Aldehyde Dehydrogenases Reveal Structural and Functional Roles of K+-Binding Sites

Many aldehyde dehydrogenases (ALDHs) have potential potassium-binding sites of as yet unknown structural or functional roles. To explore possible K+-specific effects, we performed comparative structural studies on the tetrameric betaine aldehyde dehydrogenase from Pseudomonas aeruginosa (PaBADH) and on the dimeric BADH from spinach (SoBADH), whose activities are K+-dependent and K+-independent, respectively, although both enzymes contain potassium-binding sites. Size exclusion chromatography, dynamic light scattering, far- and near-UV circular dichroism, and extrinsic fluorescence results indicated that in the absence of K+ ions and at very low ionic strength, PaBADH remained tetrameric but its tertiary structure was significantly altered, accounting for its inactivation, whereas SoBADH formed tetramers that maintained the native tertiary structure. The recovery of PaBADH native tertiary-structure was hyperbolically dependent on KCl concentration, indicating potassium-specific structuring effects probably arising from binding to a central-cavity site present in PaBADH but not in SoBADH. K+ ions stabilized the native structure of both enzymes against thermal denaturation more than did tetraethylammonium (TEA+) ions. This indicated specific effects of potassium on both enzymes, particularly on PaBADH whose apparent T m values showed hyperbolical dependence on potassium concentration, similar to that observed with the tertiary structure changes. Interestingly, we also found that thermal denaturation of both enzymes performed in low ionic-strength buffers led to formation of heat-resistant, inactive soluble aggregates that retain 80% secondary structure, have increased β-sheet content and bind thioflavin T. These structured aggregates underwent further thermal-induced aggregation and precipitation when the concentrations of KCl or TEACl were raised. Given that PaBADH and SoBADH belong to different ALDH families and differ not only in amino acid composition but also in association state and surface electrostatic potential, the formation of this kind of β-sheet pre-fibrillar aggregates, not described before for any ALDH enzyme, appear to be a property of the ALDH fold.

High concentrations of guanidine chloride activate lactate dehydrogenase in low water media

The effect of guanidine chloride on the activity of bovine heart lactate dehydrogenase transferred to a system that was made with toluene, phospholipids, Triton X-100 and 3.8% water (v/v) was studied. The activity of the enzyme in the latter system was about 30 times lower than in standard water mixtures. In the low water system, 1.5 and 2.0 M guanidine chloride increased the activity by approximately 20 times. These concentrations of guanidine chloride caused complete inactivation of the enzyme in conventional water systems. The activating effect of the denaturant was independent of enzyme concentration. It is suggested that the increase in activity produced by guanidine chloride was due to a facilitation of the protein-solvent interactions that operate in a catalytic cycle.

Exploring interfacial water trapping in protein-ligand complexes with multithermal titration calorimetry

In this work, we examine the hypothesis about how trapped water molecules at the interface between triosephosphate isomerase (TIM) and either of two phosphorylated inhibitors, 2-phosphoglycolate (2PG) or phosphoglycolohydroxamate (PGH), can explain the anomalous highly negative binding heat capacities (ΔCp,b) of both complexes, TIM-2PG and TIM-PGH. We performed fluorimetric titrations of the enzyme with PGH inhibitor under osmotic stress conditions, using various concentrations of either osmolyte: sucrose, ethylene glycol or glycine betaine. We also analyze the binding processes under various stressor concentrations using a novel calorimetric methodology that allows ΔCp,b determinations in single experiments: Multithermal Titration Calorimetry. The binding constant of the TIM-PGH complex decreased gradually with the concentration of all osmolytes, but at diverse extents depending on the osmolyte nature. According to the osmotic stress theory, this decrease indicates that the number of water molecules associated with the enzyme increases with inhibitor binding, i.e. some solvent molecules became trapped. Additionally, the binding heat capacities became less negative at higher osmolyte concentrations, their final values depending on the osmolyte. These effects were also observed in the TIM-2PG complex using sucrose as stressor. Our results strongly suggest that some water molecules became immobilized when the TIM-inhibitor complexes were formed. A computational analysis of the hydration state of the binding site of TIM in both its free state and its complexed form with 2PG or PGH, based on molecular dynamics (MD) simulations in explicit solvent, showed that the binding site effectively immobilized additional water molecules after binding these inhibitors.