Engineering a Promiscuous Tautomerase into a More Efficient Aldolase for Self‐Condensations of Linear Aliphatic Aldehydes

The enzyme 4‐oxalocrotonate tautomerase (4‐OT) from Pseudomonas putida mt‐2 takes part in a catabolic pathway for aromatic hydrocarbons, where it catalyzes the conversion of 2hydroxyhexa‐2,4‐dienedioate into 2‐oxohexa‐3‐enedioate. This tautomerase can also promiscuously catalyze carbon–carbon bond‐forming reactions, including various types of aldol reactions, by using its amino‐terminal proline as a key catalytic residue. Here, we used systematic mutagenesis to identify two hotspots in 4‐OT (Met45 and Phe50) at which single mutations give marked improvements in aldolase activity for the self‐condensation of propanal. Activity screening of a focused library in which these two hotspots were varied led to the discovery of a 4‐OT variant (M45Y/F50V) with strongly enhanced aldolase activity in the self‐condensation of linear aliphatic aldehydes, such as acetaldehyde, propanal, and butanal, to yield α,β‐unsaturated aldehydes. With both propanal and benzaldehyde, this double mutant, unlike the previously constructed single mutant F50A, mainly catalyzes the self‐condensation of propanal rather than the cross‐condensation of propanal and benzaldehyde, thus indicating that it indeed has altered substrate specificity. This variant could serve as a template to create new biocatalysts that lack dehydration activity and possess further enhanced aldolase activity, thus enabling the efficient enzymatic self‐coupling of aliphatic aldehydes.     

Evidence for the Formation of an Enamine Species during Aldol and Michael‐type Addition Reactions Promiscuously Catalyzed by 4‐Oxalocrotonate Tautomerase

The enzyme 4‐oxalocrotonate tautomerase (4‐OT), which has a catalytic N‐terminal proline residue (Pro1), can promiscuously catalyze various carbon–carbon bond‐forming reactions, including aldol condensation of acetaldehyde with benzaldehyde to yield cinnamaldehyde, and Michael‐type addition of acetaldehyde to a wide variety of nitroalkenes to yield valuable γ‐nitroaldehydes. To gain insight into how 4‐OT catalyzes these unnatural reactions, we carried out exchange studies in D₂O, and X‐ray crystallography studies. The former established that H–D exchange within acetaldehyde is catalyzed by 4‐OT and that the Pro1 residue is crucial for this activity. The latter showed that Pro1 of 4‐OT had reacted with acetaldehyde to give an enamine species. These results provide evidence of the mechanism of the 4‐OT‐catalyzed aldol and Michael‐type addition reactions in which acetaldehyde is activated for nucleophilic addition by Pro1‐dependent formation of an enamine intermediate.     

Biocatalytic and Structural Properties of a Highly Engineered Halohydrin Dehalogenase

Two highly engineered halohydrin dehalogenase variants were characterized in terms of their performance in dehalogenation and epoxide cyanolysis reactions. Both enzyme variants outperformed the wild‐type enzyme in the cyanolysis of ethyl (S)‐3,4‐epoxybutyrate, a conversion yielding ethyl (R)‐4‐cyano‐3‐hydroxybutyrate, an important chiral building block for statin synthesis. One of the enzyme variants, HheC2360, displayed catalytic rates for this cyanolysis reaction enhanced up to tenfold. Furthermore, the enantioselectivity of this variant was the opposite of that of the wild‐type enzyme, both for dehalogenation and for cyanolysis reactions. The 37‐fold mutant HheC2360 showed an increase in thermal stability of 8 °C relative to the wild‐type enzyme. Crystal structures of this enzyme were elucidated with chloride and ethyl (S)‐3,4‐epoxybutyrate or with ethyl (R)‐4‐cyano‐3‐hydroxybutyrate bound in the active site. The observed increase in temperature stability was explained in terms of a substantial increase in buried surface area relative to the wild‐type HheC, together with enhanced interfacial interactions between the subunits that form the tetramer. The structures also revealed that the substrate binding pocket was modified both by substitutions and by backbone movements in loops surrounding the active site. The observed changes in the mutant structures are partly governed by coupled mutations, some of which are necessary to remove steric clashes or to allow backbone movements to occur. The importance of interactions between substitutions suggests that efficient directed evolution strategies should allow for compensating and synergistic mutations during library design.     

Poly(Ethylene Glycol)‐Supported Proline: A Versatile Catalyst for the Enantioselective Aldol and Iminoaldol Reactions

(2S,4R)‐4‐Hydroxyproline has been anchored to the monomethyl ether of poly(ethylene glycol), M_W 5000, by means of a succinate spacer to afford a soluble, polymer‐supported catalyst (PEG‐Pro) for enantioselective aldol and iminoaldol condensation reactions. This organic catalyst can be considered as a minimalistic version of a type I aldolase enzyme, with the polymer chain replacing the enzyme's peptide backbone, and the proline residue acting as the enzyme's active site. In the presence of PEG‐Pro (0.25–0.35 mol equiv.), acetone reacted with enolizable and non‐enolizable aldehydes and imines to afford β‐ketols and β‐aminoketones in good yield and high enantiomeric excess (ee), comparable to those obtained using non‐supported proline derivatives as the catalysts. Extension of the PEG‐Pro‐promoted condensation to hydroxyacetone as the aldol donor opened an access to synthetically relevant anti‐α,β‐dihydroxyketones and syn‐α‐hydroxy‐β‐aminoketones, that were obtained in moderate to good yields, and good to high diastereo‐ and enantioselectivity. Exploiting its solubility properties, the PEG‐Pro catalyst was easily recovered and recycled to promote all of the above‐mentioned reactions, that occurred in slowly diminishing yields but virtually unchanged ee's.     

Catalyzed gasification of active carbon by oxygen: influence of catalyst type,temperature, oxygen partial pressure and particle size

A thermogravimetric study of the oxygen gasification of a commercial active carbon using Co, Ni and Cu as catalysts has been carried out. This follows a previous study of the influence of the particle size, gas volumetric flow rate, initial sample weight temperature and oxygen partial pressure an non‐catalytic gasification. The results show that for T < 500 °C; P₀₂ < 1 atm; D _p < 0.4 mm, Q _v > 50 cm³ min⁻¹ and M _o < 75 mg the gasification of the active carbon with oxygen is controlled by the chemical reaction. The presence of a catalyst increases in all cases the reaction rate, both temperature and catalyst concentration showing a very positive influence. The activity of the different catalysts follows the order: Co > Cu > Ni. A kinetic study applied to both types of gasification has allowed the activation energy and the reaction order with respect to oxygen and the catalyst to be determined. In all cases both the activation energy and the Arrhenius pre‐exponential factor are lower in the catalytic gasification. This brings about the existence of an isokinetic point for the catalytic and the non‐catalytic processes, which were also determined. © 2000 Society of Chemical Industry     

4‐Hydroxy‐3‐methyl‐6‐(1‐methyl‐2‐oxoalkyl)pyran‐2‐one Synthesis by a Type III Polyketide Synthase from Rhodospirillum centenum

The purple photosynthetic bacterium Rhodospirillum centenum has a putative type III polyketide synthase gene (rpsA). Although rpsA was known to be transcribed during the formation of dormant cells, the reaction catalyzed by RpsA was unknown. Thus we examined the RpsA reaction in vitro, using various fatty acyl‐CoAs with even numbers of carbons as starter substrates. RpsA produced tetraketide pyranones as major compounds from one C_10–14 fatty acyl‐CoA unit, one malonyl‐CoA unit and two methylmalonyl‐CoA units. We identified these products as 4‐hydroxy‐3‐methyl‐6‐(1‐methyl‐2‐oxoalkyl)pyran‐2‐ones by NMR analysis. RpsA is the first bacterial type III PKS that prefers to incorporate two molecules of methylmalonyl‐CoA as the extender substrate. In addition, in vitro reactions with ¹³C‐labeled malonyl‐CoA revealed that RpsA produced tetraketide 6‐alkyl‐4‐hydroxy‐1,5‐dimethyl‐2‐oxocyclohexa‐3,5‐diene‐1‐carboxylic acids from C_14–20 fatty acyl‐CoAs. This class of compounds is likely synthesized through aldol condensation induced by methine proton abstraction. No type III polyketide synthase that catalyzes this reaction has been reported so far. These two unusual features of RpsA extend the catalytic functions of the type III polyketide synthase family.     

Investigation of Structural Determinants for the Substrate Specificity in the Zinc‐Dependent Alcohol Dehydrogenase CPCR2 from Candida parapsilosis

Zinc‐dependent alcohol dehydrogenases (ADHs) are a class of enzymes applied in different biocatalytic processes ranging from lab to industrial scale. However, one drawback is the limited substrate range, necessitating a whole array of different ADHs for the relevant substrate classes. In this study, we investigated structural determinants of the substrate spectrum in the zinc‐dependent ADH carbonyl reductase 2 from Candida parapsilosis (CPCR2), combining methods of mutational analysis with in silico substrate docking. Assigned active site residues were genetically randomized, and the resulting mutant libraries were screened with a selection of challenging carbonyl substrates. Three variants (C57A, W116K, and L119M) with improved activities toward different substrates were detected at neighboring positions in the active site. Thus, all possible combinations of the mutations were generated and characterized for their substrate specificity, yielding several improved variants. The most interesting were a C57A variant, with a 27‐fold increase in specific activity for 4′‐acetamidoacetophenone, and the double mutant CPCR2 B16‐(C57A, L119M), with a 45‐fold improvement in the k_cat?K_M^?1 value. The obtained variants were further investigated by in silico docking experiments. The results indicate that the mentioned residues are structural determinants of the substrate specificity of CPCR2, being major players in the definition of the active site. Comparison of these results with closely related enzymes suggests that these might even be transferred to other ADHs.     

Detailed Structure–Function Correlations of Bacillus subtilis Acetolactate Synthase

Isobutanol is deemed to be a next‐generation biofuel and a renewable platform chemical. 1 Non‐natural biosynthetic pathways for isobutanol production have been implemented in cell‐based and in vitro systems with Bacillus subtilis acetolactate synthase (AlsS) as key biocatalyst. 2 – 6 AlsS catalyzes the condensation of two pyruvate molecules to acetolactate with thiamine diphosphate and Mg²⁺ as cofactors. AlsS also catalyzes the conversion of 2‐ketoisovalerate into isobutyraldehyde, the immediate precursor of isobutanol. Our phylogenetic analysis suggests that the ALS enzyme family forms a distinct subgroup of ThDP‐dependent enzymes. To unravel catalytically relevant structure‐function relationships, we solved the AlsS crystal structure at 2.3 Å in the presence of ThDP, Mg²⁺ and in a transition state with a 2‐lactyl moiety bound to ThDP. We supplemented our structural data by point mutations in the active site to identify catalytically important residues.     

Modification of Substrate Specificity Resulting in an Epoxide Hydrolase with Shifted Enantiopreference for (2,3‐Epoxypropyl)benzene

Random mutagenesis targeted at hotspots of noncatalytic active‐site residues of potato epoxide hydrolase StEH1 combined with an enzyme‐activity screen allowed the isolation of enzyme variants displaying altered enantiopreference in the catalyzed hydrolysis of (2,3‐epoxypropyl)benzene. The wild‐type enzyme favored the S enantiomer with a ratio of 2.5:1, whereas the variant displaying the most radical functional changes showed a 15:1 preference for the R enantiomer. This mutant had accumulated four substitutions distributed over two out of four mutated hotspots: W106L, L109Y, V141K, and I151V. The underlying causes of the enantioselectivity were a decreased catalytic efficiency in the catalyzed hydrolysis of the S enantiomer combined with retained activity with the R enantiomer. The results demonstrate the feasibility of molding the stereoselectivity of this biocatalytically relevant enzyme.     

Changing the regioselectivity of a P450 from C15 to C11 hydroxylation of progesterone

CYP106A2 is known as a 15β‐hydroxylase, but also shows minor 11α‐hydroxylase activity for progesterone. 11α‐Hydroxyprogesterone is an important pharmaceutical compound with anti‐androgenic and blood‐pressure‐regulating activity. This work therefore focused on directing the regioselectivity of the enzyme towards hydroxylation at position 11 in the C ring of the steroid through a combination of saturation mutagenesis and rational site‐directed mutagenesis. With the aid of data from a homology model of CYP106A2 containing docked progesterone, together with site‐directed mutagenesis of active‐site residues (Lisurek et al. ChemBioChem 2008 , 9, 1439–1449), a saturation mutagenesis library at positions A395 and G397 was created. Screening of the library identified the mutants A395I and A395W/G397K as having 11α‐hydroxylase activities 8.9 and 11.5 times higher than that of the wild type (WT). In the next step, additional mutations were integrated by a rational site‐directed mutagenesis approach to increase the catalytic efficiency. Of the 40 candidates analyzed, the mutants A106T/A395I, A106T/A395I/R409L, and T89N/A395I turned out to display increased 11α‐hydroxylase selectivities and activities relative to the WT (14.3‐, 12.6‐, and 11.8‐fold increases in selectivity and 39.3‐, 108‐, and 24.4‐ in k_cat/K_m). In the last step of the study, the best mutants were applied in a whole‐cell biotransformation. In these experiments the production (percentage) of 15β‐hydroxyprogesterone decreased from 50.4 % (wild type) to 4.8 % (mutant T89N/A395I), whereas that of 11α‐hydroxyprogesterone increased from 27.7 to 80.9 %, thus demonstrating an impressive regioselectivity.     

Increasing the Reaction Rate of Hydroxynitrile Lyase from Hevea brasiliensis toward Mandelonitrile by Copying Active Site Residues from an Esterase that Accepts Aromatic Esters

The natural substrate of hydroxynitrile lyase from rubber tree (HbHNL, Hevea brasiliensis) is acetone cyanohydrin, but synthetic applications usually involve aromatic cyanohydrins such as mandelonitrile. To increase the activity of HbHNL toward this unnatural substrate, we replaced active site residues in HbHNL with the corresponding ones from esterase SABP2 (salicylic acid binding protein 2). Although this enzyme catalyzes a different reaction (hydrolysis of esters), its natural substrate (methyl salicylate) contains an aromatic ring. Three of the eleven single‐amino‐acid‐substitution variants of HbHNL reacted more rapidly with mandelonitrile. The best was HbHNL‐L121Y, with a k_cat 4.2 times higher and high enantioselectivity. Site‐saturation mutagenesis at position 121 identified three other improved variants. We hypothesize that the smaller active site orients the aromatic substrate more productively.     

Redesign of the Phosphate Binding Site of L‐Rhamnulose‐ 1‐Phosphate Aldolase towards a Dihydroxyacetone Dependent Aldolase

The aldol addition of unphosphorylated dihydroxyacetone (DHA) to aldehydes catalyzed by L ‐rhamnulose‐1‐phosphate aldolase (RhuA), a dihydroxyacetone phosphate‐dependent aldolase, is reported. Moreover, a single point mutation in the phosphate binding site of the RhuA wild type, that is, substitution of aspartate for asparagine at position N29, increased by 3‐fold the of aldol addition reactions of DHA to other aldehyde acceptors rather than the natural L ‐lactaldehyde. The RhuA N29D mutant modified the optimum enzyme design for the natural substrate and changed its catalytic properties making the aldolase more versatile to other aldol additions of DHA.     

The cooperative effect between active site ionized groups and water desolvation controls the alteration of acid/base catalysis in serine proteases

What is the driving force that alters the catalytic function of His57 in serine proteases between general base and general acid in each step along the enzymatic reaction? The stable tetrahedral complexes (TC) of chymotrypsin with trifluoromethyl ketone transition state analogue inhibitors are topologically similar to the catalytic transition state. Therefore, they can serve as a good model to study the enzyme catalytic reaction. We used DFT quantum mechanical calculations to analyze the effect of solvation and of polar factors in the active site of chymotrypsin on the pK_a of the catalytic histidine in FE (the free enzyme), EI (the noncovalent enzyme inhibitor complex), and TC. We demonstrated that the acid/base alteration is controlled by the charged groups in the active site—the catalytic Asp102 carboxylate and the oxyanion. The effect of these groups on the catalytic His is modulated by water solvation of the active site.     

Directed evolution of subtilisin E into a highly active and guanidinium chloride- and sodium dodecylsulfate-tolerant protease

Proteases have niche applications in diagnostic kits that use cell lysis and thereby require high resistance towards chaotropic salts and detergents, such as guanidinium chloride (GdmCl) and sodium dodecylsulfate (SDS). Subtilisin E, a well‐studied serine protease, was selected to be re‐engineered by directed evolution into a “chaophilic” protease that would be resistance to GdmCl and SDS, for application in diagnostic kits. In three iterative rounds of directed evolution, variant SeSaM1–5 (S62I/A153V/G166S/I205V) was generated, with improved activity (330 %) and increased half life in 1 M GdmCl (<2 min to 4.7 h) or in 0.5 % SDS (<2 min to 2.7 h). Saturation mutagenesis at each site in the wild‐type subtilisin E revealed that positions 62 and 166 were mainly responsible for increased activity and stability. A double mutant, M2 (S62I/G166M), generated by combination of the best single mutations showed significantly improved kinetic constants; in 2 M GdmCl the K_m value decreased (29‐fold) from 7.31 to 0.25 mM , and the k_cat values increased (fourfold) from 15 to 61 s^?1. The catalytic efficiency, k_cat/K_m, improved dramatically (GdmCl: 247 mM ^?1 s^?1 (118‐fold); SDS, 179 mM ^?1 s^?1 (13‐fold)). In addition, the SeSaM1–5 variant showed higher stability in 2.0 % SDS when compared to the wild‐type (t_1/2 54.8 min (>27‐fold)). Finally, molecular dynamics simulations of the wild‐type subtilisin E showed that Gdm⁺ ions could directly interact with active site residues, thereby probably limiting access of the substrate to the catalytic centre.     

Reaction Mechanism of Prephenate Dehydrogenase from the Alternative Tyrosine Biosynthesis Pathway in Plants

Unlike metazoans, plants, bacteria, and fungi retain the enzymatic machinery necessary to synthesize the three aromatic amino acids l ‐phenylalanine, l ‐tyrosine, and l ‐tryptophan de novo. In legumes, such as soybean, alfalfa, and common bean, prephenate dehydrogenase (PDH) catalyzes the tyrosine‐insensitive biosynthesis of 4‐hydroxyphenylpyruvate, a precursor to tyrosine. The three‐dimensional structure of soybean PDH1 was recently solved in complex with the NADP⁺ cofactor. This structure allowed for the identification of both the cofactor‐ and ligand‐binding sites. Here, we present steady‐state kinetic analysis of twenty site‐directed active‐site mutants of soybean (Glycine max) PDH compared to wild‐type. Molecular docking of the substrate, prephenate, into the active site of the enzyme revealed its potential interactions with the active site residues and made a case for the importance of each residue in substrate recognition and/or catalysis, most likely through transition state stabilization. Overall, these results suggested that the active site of the enzyme is highly sensitive to any changes, as even subtle alterations substantially reduced the catalytic efficiency of the enzyme.     

A Single Mutation Increases the Activity and Stability of Pectobacterium carotovorum Nitrile Reductase

Nitrile reductases are considered to be promising and environmentally benign nitrile‐reducing biocatalysts to replace traditional metal catalysts. Unfortunately, the catalytic efficiencies of the nitrile reductases reported so far are very low. To date, all attempts to increase the catalytic activity of nitrile reductases by protein engineering have failed. In this work, we successfully increased the specific activity of a nitrile reductase from Pectobacterium carotovorum from 354 to 526 U g_prot^?1 by engineering the substrate binding pocket; moreover, the thermostability was also improved (≈2‐fold), showing half‐lives of 140 and 32 h at 30 and 40 °C, respectively. In the bioreduction of 2‐amino‐5‐cyanopyrrolo[2,3‐d]pyrimidin‐4‐one (preQ₀) to 2‐amino‐5‐aminomethylpyrrolo[2,3‐d]pyrimidin‐4‐one (preQ₁), the variant was advantageous over the wild‐type enzyme with a higher reaction rate and complete conversion of the substrate within a shorter period. Homology modeling and docking analysis revealed some possible origins of the increased activity and stability. These results establish a solid basis for future engineering of nitrile reductases to increase the catalytic efficiency further, which is a prerequisite for applying these novel biocatalysts in synthetic chemistry.     

Targeting tumour proliferation with a small-molecule inhibitor of AICAR transformylase homodimerization

Aminoimidazole carboxamide ribonucleotide transformylase/ inosine monophosphate cyclohydrolase (ATIC) is a bifunctional homodimeric enzyme that catalyzes the last two steps of de novo purine biosynthesis. Homodimerization of ATIC, a protein–protein interaction with an interface of over 5000 Ų, is required for its aminoimidazole carboxamide ribonucleotide (AICAR) transformylase activity, with the active sites forming at the interface of the interacting proteins. Here, we report the development of a small‐molecule inhibitor of AICAR transformylase that functions by preventing the homodimerization of ATIC. The compound is derived from a previously reported cyclic hexapeptide inhibitor of AICAR transformylase (with a K_i of 17 μM ), identified by high‐throughput screening. The active motif of the cyclic peptide is identified as an arginine‐tyrosine dipeptide, a capped analogue of which inhibits AICAR transformylase with a K_i value of 84 μM . A library of nonnatural analogues of this dipeptide was designed, synthesized, and assayed. The most potent compound inhibits AICAR transformylase with a K_i value of 685 nM , a 25‐fold improvement in activity from the parent cyclic peptide. The potential for this AICAR transformylase inhibitor in cancer therapy was assessed by studying its effect on the proliferation of a model breast cancer cell line. Using a nonradioactive proliferation assay and live cell imaging, a dose‐dependent reduction in cell numbers and cell division rates was observed in cells treated with our ATIC dimerization inhibitor.     

Chaperone Activity of Bicyclic Nojirimycin Analogues for Gaucher Mutations in Comparison with N‐(n‐nonyl)Deoxynojirimycin

Gaucher disease (GD), the most prevalent lysosomal storage disorder, is caused by mutations of lysosomal β‐glucosidase (acid β‐Glu, β‐glucocerebrosidase); these mutations result in protein misfolding. Some inhibitors of this enzyme, such as the iminosugar glucomimetic N‐(n‐nonyl)‐1‐deoxynojirimycin (NN‐DNJ), are known to bind to the active site and stabilize the proper folding for the catalytic form, acting as “chemical chaperones” that facilitate transport and maturation of acid β‐Glu. Recently, bicyclic nojirimycin (NJ) analogues with structure of sp² iminosugars were found to behave as very selective, competitive inhibitors of the lysosomal β‐Glu. We have now evaluated the glycosidase inhibitory profile of a series of six compounds within this family, namely 5‐N,6‐O‐(N′‐octyliminomethylidene‐NJ (NOI‐NJ), the 6‐thio and 6‐amino‐6‐deoxy derivatives (6S‐NOI‐NJ and 6N‐NOI‐NJ) and the corresponding galactonojirimycin (GNJ) counterparts (NOI‐GNJ, 6S‐NOI‐GNJ and 6N‐NOI‐GNJ), against commercial as well as lysosomal glycosidases. The chaperone effects of four selected candidates (NOI‐NJ, 6S‐NOI‐NJ, 6N‐NOI‐NJ, and 6S‐NOI‐GNJ) were further evaluated in GD fibroblasts with various acid β‐Glu mutations. The compounds showed enzyme enhancement on human fibroblasts with N188S, G202R, F213I or N370S mutations. The chaperone effects of the sp² iminosugar were generally stronger than those observed for NN‐DNJ; this suggests that these compounds are promising candidates for clinical treatment of GD patients with a broad range of β‐Glu mutations, especially for neuronopathic forms of Gaucher disease.     

Functional Elastomers via Sequential Selective Diene Copolymerization/Hydrophosphorylation Catalysis

An effective preparation of new tailor‐made macromolecular materials via the combination of two fully atom‐efficient catalytic transformations is reported. First, new isoprene‐co‐1,3,7‐octatriene [P(IP‐co‐OT)] copolymers with controlled composition (2–21 mol% triene incorporated), molecular weight (M_n=23–102,000 g mol⁻¹) and microstructure (1,4‐cis rich) have been prepared using the homogeneous Ziegler–Natta catalyst system composed of diallylneodynium chloride, magnesium chloride, tetrahydrofuran and methylaluminoxane [Nd(allyl)₂Cl(MgCl₂)₂(THF)₄/MAO]. Next, the pendant vinyl moieties in those P(IP‐co‐OT) copolymers have been selectively transformed into phosphonate groups by hydrophosphorylation in the presence of homogeneous rhodium phosphine catalysts. The latter hydrophosphorylation reaction has been optimized in terms of catalytic activity and selectivity, to define the conditions for an effective and safe procedure that does not affect the macromolecular architecture. All polymer materials have been microstructurally analyzed by ¹H, ¹³C, and ³¹P NMR spectroscopy, to diagnose the catalyst selectivities in the copolymerization and hydrophosphorylation processes.     

The Inactivation Mechanism of Human Group IIA Phospholipase A2 by Scalaradial

Secretory phospholipases A₂ (sPLA₂s) are implicated in the pathogenesis of several inflammation diseases, such as rheumatoid arthritis, septic shock, psoriasis, and asthma. Thus, an understanding of their inactivation mechanisms could be useful for the development of new classes of chemical selective inhibitors. In the marine environment, several bioactive terpenoids possess interesting anti‐inflammatory activity, often through covalent and/or noncovalent inactivation of sPLA₂. Herein, we report the molecular mechanism of human group IIA phospholipase A₂ (sPLA₂‐IIA) inactivation by Scalaradial (SLD), a marine 1,4‐dialdehyde terpenoid isolated from the sponge Cacospongia mollior and endowed with a significant anti‐inflammatory profile. Our results have been collected by a combination of biochemical approaches, advanced mass spectrometry, surface plasmon resonance, and molecular modeling. These suggest that SLD acts as a competitive inhibitor. Indeed, the sPLA₂‐IIA inactivation process seems to be driven by the noncovalent recognition process of SLD in the enzyme active site and by chelation of the catalytic calcium ion. In contrast, covalent modification of the enzyme by the SLD dialdehyde moiety emerges as only a minor side event in the ligand–enzyme interaction. These results could be helpful for the rational design of new PLA₂ inhibitors that would be able to selectively target the enzyme active site.