Homology modeling of 5-lipoxygenase and hints for better inhibitor design

Lipoxygenases (LOXs) are a group of enzymes involved in the oxygenation of polyunsaturated fatty acids. Among these 5-lipoxygenase (5-LOX) is the key enzyme leading to the formation of pharmacologically important leukotrienes and lipoxins, the mediators of inflammatory and allergic disorders. In view of close functional similarity to mammalian lipoxygenase, potato 5-LOX is used extensively. In this study, the homology modeling technique has been used to construct the structure of potato 5-LOX. The amino acid sequence identity between the target protein and sequence of template protein 1NO3 (soybean LOX-3) searched from NCBI protein BLAST was 63%. Based on the template structure, the protein model was constructed by using the Homology program in InsightII. The protein model was briefly refined by energy minimization steps and validated using Profile-3D, ERRAT and PROCHECK. The results showed that 99.3% of the amino acids were in allowed regions of Ramachandran plot, suggesting that the model is accurate and its stereochemical quality good. Like all LOXs, 5-LOX also has a two-domain structure, the small N-terminal beta-barrel domain and a larger catalytic domain containing a single atom of non-heme iron coordinating with His525, His530, His716 and Ile864. Asn720 is present in the fifth coordination position of iron. The sixth coordination position faces the open cavity occupied here by the ligands which are docked. Our model of the enzyme is further validated by examining the interactions of earlier reported inhibitors and by energy minimization studies which were carried out using molecular mechanics calculations. Four ligands, nordihydroguaiaretic acid (NDGA) having IC(50) of 1.5 microM and analogs of benzyl propargyl ethers having IC(50) values of 760 microM, 45 microM, and no inhibition respectively were selected for our docking and energy minimization studies. Our results correlated well with the experimental data reported earlier, which proved the quality of the model. This model generated can be further used for the design and development of more potent 5-LOX inhibitors.     

Control of oxygenation in lipoxygenase and cyclooxygenase catalysis

Lipoxygenases (LOX) and cyclooxygenases (COX) react an achiral polyunsaturated fatty acid with oxygen to form a chiral peroxide product of high regio- and stereochemical purity. Both enzymes employ free radical chemistry reminiscent of hydrocarbon autoxidation but execute efficient control during catalysis to form a specific product over the multitude of isomers found in the nonenzymatic reaction. Exactly how both dioxygenases achieve this positional and stereo control is far from clear. We present four mechanistic models, not mutually exclusive, that could account for the specific reactions of molecular oxygen with a fatty acid in the LOX or COX active site.     

Chemical Probing of the Human Sirtuin 5 Active Site Reveals Its Substrate Acyl Specificity and Peptide‐Based Inhibitors

Sirtuins are NAD⁺‐dependent deacetylases acting as sensors in metabolic pathways and stress response. In mammals there are seven isoforms. The mitochondrial sirtuin 5 is a weak deacetylase but a very efficient demalonylase and desuccinylase; however, its substrate acyl specificity has not been systematically analyzed. Herein, we investigated a carbamoyl phosphate synthetase 1 derived peptide substrate and modified the lysine side chain systematically to determine the acyl specificity of Sirt5. From that point we designed six potent peptide‐based inhibitors that interact with the NAD⁺ binding pocket. To characterize the interaction details causing the different substrate and inhibition properties we report several X‐ray crystal structures of Sirt5 complexed with these peptides. Our results reveal the Sirt5 acyl selectivity and its molecular basis and enable the design of inhibitors for Sirt5.     

Modulation of hypericin photodynamic therapy by pretreatment with 12 various inhibitors of arachidonic acid metabolism in colon adenocarcinoma HT-29 cells

One proposal to increase the efficiency of photodynamic therapy (PDT) is to accompany photosensitization with other treatment modalities, including modulation of arachidonic acid (AA) metabolism. The aim of this study was to evaluate the effectiveness of a combined modality approach employing 48 and 24 h pretreatment with various inhibitors of lipoxygenase (LOX; nordihydroguaiaretic acid, esculetin, AA-861, MK-886 and baicalein), cyclooxygenase (COX; diclofenac, flurbiprofen, ibuprofen, indomethacin, SC-560 and rofecoxib) and cytochrome P450-monooxygenase (proadifen) pathways, followed by hypericin-mediated PDT. Cytokinetic parameters like MTT assay, adherent and floating cell numbers, viability and cell cycle distribution analysis were examined 24 h after hypericin activation. Pretreatment of human colon cancer cells HT-29 prior to PDT with 5-LOX inhibitor MK-886 as well as 5, 12-LOX and 12-LOX inhibitors (esculetin and baicalein, respectively) resulted in significant and dose-dependent effects on all parameters tested. Pretreatment with diclofenac, flurbiprofen, ibuprofen and indomethacin, the nonspecific COX inhibitors, promoted hypericin-mediated PDT, but these effects were probably COX-independent. In contrast, application of SC-560 and rofecoxib, specific inhibitors of COX-1 and COX-2, respectively, attenuated PDT. Inhibition of P450 monooxygenase with proadifen implied also the significance of this metabolic pathway in cell survival and cell resistance to hypericin photocytotoxicity. In conclusion, our results testify that application of diverse inhibitors of AA metabolism may have different consequences on cellular response to hypericin-mediated PDT and that some of them could be considered for potentiation of PDT.     

Regulation of apoptosis through arachidonate cascade in mammalian cells

The arachidonate cascade includes the cyclooxygenase (COX) pathway to form prostanoids and the lipoxygenase (LOX) pathway to generate several oxygenated fatty acids, collectively called eicosanoids. Eicosanoids are suggested to play a dual role in regulating cell survival and apoptosis in various types of cells through an unknown mechanism. We found apoptosis in cultured Madin-Darby canine kidney (MDCK) cells treated with 12-O-tetradecanoylphorbol β-acetate (TPA), a potent tumor promoter, and nordihydroguaiaretic acid (NDGA), a LOX inhibitor. The effect of TPA was synergistically stimulated along with NDGA. Aspirin, a COX inhibitor, was not effective. The target of NDGA might be different from the mechanism involving a LOX activity in some kinds of carcinoma cells because the increased expression of 12-LOX was not detected in MDCK cells treated with TPA. Caspase and poly(ADP-ribose) metabolites were found to be involved in the signal transduction pathway of the TPA- and NDGA-induced apoptosis in MDCK cells. Alternatively, hydrogen peroxide-induced apoptosis was not affected by NDGA. Thus, the TPA-induced response involved the mechanism independent of the oxidative stress. Obesity is a risk factor for severe diseases including noninsulin-dependent diabetes and atherosclerosis characterized by the changes of cell properties of adipocytes. We found that conjugated linolenic acid from bitter gourd was able to induce apoptosis in mouse preadipogenic 3T3-L1 cells. The findings provide the potential use of conjugated fatty acids to regulate obesity.     

Replacement of an Indole Scaffold Targeting Human 15‐Lipoxygenase‐1 Using Combinatorial Chemistry

Human 15‐lipoxygenase‐1 (15‐LOX‐1) belongs to the class of lipoxygenases, which catalyze oxygenation of polyunsaturated fatty acids, such as arachidonic and linoleic acid. Recent studies have shown that 15‐LOX‐1 plays an important role in physiological processes linked to several diseases such as airway inflammation disease, coronary artery disease, and several types of cancer such as rectal, colon, breast and prostate cancer. In this study, we aimed to extend the structural diversity of 15‐LOX‐1 inhibitors, starting from the recently identified indolyl core. In order to find new scaffolds, we employed a combinatorial approach using various aromatic aldehydes and an aliphatic hydrazide tail. This scaffold‐hopping study resulted in the identification of the 3‐pyridylring as a suitable replacement of the indolyl core with an inhibitory activity in the micromolar range (IC₅₀=16±6 μm ) and a rapid and efficient structure–activity relationship investigation.     

Substrate binding to mammalian 15-lipoxygenase

Lipoxygenases (LOs) are implicated in the regulation of metabolic processes and in several human diseases. Revealing their exact role is hindered by an incomplete understanding of their activity, including substrate specificity and substrate alignment in the active site. Recently, it has been proposed that the change in substrate specificity for arachidonic acid (AA) or linoleic acid (LA) could be part of an auto-regulatory mechanism related to cancer grow. Kinetic differences between reactions of 15-hLO with AA and LA have also led to the suggestion that the two substrates could present mechanistic differences. In the absence of a crystal structure for the substrate:15-LO complex, here we present an atomic-level study of catalytically competent binding modes for LA to rabbit 15-LO (15-rLO-1) and compare the results to our previous work on AA. Docking calculations, molecular dynamics simulations, re-docking and cross-docking calculations are all used to analyze the differences and similarities between the binding modes of the two substrates. Interestingly, LA seems to adapt more easily to the enzyme structure and differs from AA on some dynamical aspects that could introduce kinetic differences, as observed experimentally. Still, our study concludes that, despite the different chain lengths and number of insaturations between these two physiological substrates of 15-rLO-1, the enzyme seems to catalyze their hydroperoxidation by binding them with a common binding mode that leads to similar catalytically competent complexes.     

Exploring the active site of amine:pyruvate aminotransferase on the basis of the substrate structure-reactivity relationship: how the enzyme controls substrate specificity and stereoselectivity

An active site model of the amine:pyruvate aminotransferase (APA) from Vibrio fluvialis JS17 was constructed on the basis of the relationship between substrate structure and reactivity. Due to the broad substrate specificity of the APA, various amino donors (chiral and achiral amine, amino acid, and amino acid derivative) and amino acceptors (keto acid, keto ester, aldehyde, and ketone) were used to explore the active site structure. The result suggested a two-binding site model consisting of two pockets, one large (L) and the other small (S). The difference in the size of each binding pocket and strong repulsion for a carboxylate in the S pocket were key determinants to control its substrate specificity and stereoselectivity. The L pocket showed dual recognition mode for both hydrophobic and carboxyl groups as observed in the side-chain pockets of aspartate aminotransferase and aromatic aminotransferase. Comparison of the model with those of other aminotransferases revealed that the L and S pockets corresponded to carboxylate trap and side-chain pocket, respectively. The active site model successfully explains the observed substrate specificity as well as the stereoselectivity of the APA.     

Sequence-Based Prediction of Promiscuous Acyltransferase Activity in Hydrolases

Certain hydrolases preferentially catalyze acyl transfer over hydrolysis in an aqueous environment. However, the molecular and structural reasons for this phenomenon are still unclear. Herein, we provide evidence that acyltransferase activity in esterases highly correlates with the hydrophobicity of the substrate-binding pocket. A hydrophobicity scoring system developed in this work allows accurate prediction of promiscuous acyltransferase activity solely from the amino acid sequence of the cap domain. This concept was experimentally verified by systematic investigation of several homologous esterases, leading to the discovery of five novel promiscuous acyltransferases. We also developed a simple yet versatile colorimetric assay for rapid characterization of novel acyltransferases. This study demonstrates that promiscuous acyltransferase activity is not as rare as previously thought and provides access to a vast number of novel acyltransferases with diverse substrate specificity and potential applications.     

Synthesis of 11-thialinoleic acid and 14-thialinoleic acid, inhibitors of soybean and human lipoxygenases

Lipoxygenases catalyse the oxidation of polyunsaturated fatty acids and have been invoked in many diseases including cancer, atherosclerosis and Alzheimer's disease. Currently, no X-ray structures are available with substrate or substrate analogues bound in a productive conformation. Such structures would be very useful for examining interactions between substrate and active site residues. Reported here are the syntheses of linoleic acid analogues containing a sulfur atom at the 11 or 14 positions. The key steps in the syntheses were the incorporation of sulfur using nucleophilic attack of metallated alkynes on electrophilic sulfur compounds and the subsequent stereospecific tantalum-mediated reduction of the alkynylsulfide to the cis-alkenylsulfide. Kinetic assays performed with soybean lipoxygenase-1 showed that both 11-thialinoleic acid and 14-thialinoleic acid were competitive inhibitors with respect to linoleic acid with K(i) values of 22 and 35 microM, respectively. On the other hand, 11-thialinoleic acid was a noncompetitive inhibitor with respect to arachidonic acid with K(is) and K(ii) values of 48 and 36 microM, respectively. 11-Thialinoleic acid was also a noncompetitive inhibitor of human 15-lipoxygenase-1 with arachidonic acid (K(is) = 11.4 microM, K(ii) = 18.1 microM) or linoleic acid as substrate (K(is) = 20.1 microM, K(ii) = 20.0 microM), and a competitive inhibitor of human 12-lipoxygenase with arachidonic acid as substrate (K(i) = 2.5 microM). The presence of inhibitor did not change the regioselectivity of soybean lipoxygenase-1, human 12- or 15-lipoxygenase-1.     

Microsomal delta 9, delta 6 and delta 5 desaturase activities and liver membrane fatty acid profiles in alcohol-fed rats

In experimentally produced alcoholic fatty liver microsomal fatty acid composition was measured using gas chromatography. The results showed an increase in linoleic acid (18:2, n-6) and hexadecaenoic acid (22:6, n-3) and a decrease in arachidonic acid (20:4, n-6) in alcohol-fed rats. Using high performance liquid chromatographic separation of radiolabelled substrate and products, delta 9, delta 6 and delta 5 desaturase enzymes were assayed. The activity of delta 9 and delta 5 desaturase was decreased in alcohol-fed rats and delta 6 desaturase activity was similar in control and alcohol-fed groups. These results indicated there was no causal relationship between desaturase activity and membrane fatty acid changes. Increased amounts of eicosatrienoic acid (20:3, n-9) in rats fed less than 5% fat were observed in both control and alcohol-fed rats. The results indicated that essential fatty acid deficiency was not due to alcohol consumption.     

Crystal structures and catalytic mechanism of the Arabidopsis cinnamyl alcohol dehydrogenases AtCAD5 and AtCAD4

The cinnamyl alcohol dehydrogenase (CAD) multigene family in planta encodes proteins catalyzing the reductions of various phenylpropenyl aldehyde derivatives in a substrate versatile manner, and whose metabolic products are the precursors of structural lignins, health-related lignans, and various other metabolites. In Arabidopsis thaliana, the two isoforms, AtCAD5 and AtCAD4, are the catalytically most active being viewed as mainly involved in the formation of guaiacyl/syringyl lignins. In this study, we determined the crystal structures of AtCAD5 in the apo-form and as a binary complex with NADP+, respectively, and modeled that of AtCAD4. Both AtCAD5 and AtCAD4 are dimers with two zinc ions per subunit and belong to the Zn-dependent medium chain dehydrogenase/reductase (MDR) superfamily, on the basis of their overall 2-domain structures and distribution of secondary structural elements. The catalytic Zn2+ ions in both enzymes are tetrahedrally coordinated, but differ from those in horse liver alcohol dehydrogenase since the carboxyl side-chain of Glu70 is ligated to Zn2+ instead of water. Using AtCAD5, site-directed mutagenesis of Glu70 to alanine resulted in loss of catalytic activity, thereby indicating that perturbation of the Zn2+ coordination was sufficient to abolish catalytic activity. The substrate-binding pockets of both AtCAD5 and AtCAD4 were also examined, and found to be significantly different and smaller compared to that of a putative aspen sinapyl alcohol dehydrogenase (SAD) and a putative yeast CAD. While the physiological roles of the aspen SAD and the yeast CAD are uncertain, they nevertheless have a high similarity in the overall 3D structures to AtCAD5 and 4. With the bona fide CAD's from various species, nine out of the twelve residues which constitute the proposed substrate-binding pocket were, however, conserved. This is provisionally considered as indicative of a characteristic fingerprint for the CAD family.     

Understanding the Behavior of Thermomyces lanuginosus Lipase in Acylation of Pyrimidine Nucleosides Possessing 2′-Substituent

The effect of 2′-differing substituent groups (such as the H, F, Cl, Br, OCH₃) as well as various chain lengths of the acyl substrates (C6, C10, and C14) on the performance of Thermomyces lanuginosus lipase for the regioselective acylation of pyrimidine nucleoside analogs was investigated systematically for the first time. The results evidently demonstrated that changing substituents in nucleosides had a significant influence on the substrate specificity for the enzymatic reaction, possibly due to the differences in physicochemical properties of the substituents and the special shape of the substrate binding site in enzyme active region. With respect to the chain-length selectivity, the enzyme exhibited the highest 5′-regioselectivity toward the longer chain (C14) as compared to short (C6 and C10) ones. The reason might be attributed to the presence of the interaction between the acyl chain and the large hydrophobic substrate-binding pocket.     

血必净抗炎作用药效物质基础和多靶点作用效应

研究了血必净抗炎作用的药效物质基础, 并在分子层面上对复方多靶点作用进行阐释. 借助于计算机辅助药物设计技术, 构建血必净化学成分数据库, 综合应用同源模建、分子对接、药效团模型、数据库搜索等方法, 探讨其与炎症靶点5-脂氧合酶(5-LOX)、环氧合酶-2(COX-2)、IKK-2受体的相互作用关系. 血必净化学成分中与靶点5-LOX、COX-2、IKK-2结合效应较好的分别有30、36、8个分子; 有16个分子与2个或3个靶点存在作用, 其中15个分子对靶点5-LOX和COX-2有抑制作用, 迷迭香酸对3个靶点均有作用. 从分子层次上阐释了复方血必净抗炎作用的药效物质基础和多靶点作用效应, 为血必净复方的临床应用提供了科学依据; 同时, 也为寻找新型抗炎药物提供一定的参考和借鉴.     

Synthesis, cytotoxicity, cellular uptake and influence on eicosanoid metabolism of cobalt-alkyne modified fructoses in comparison to auranofin and the cytotoxic COX inhibitor Co-ASS

Propargylhexacarbonyldicobalt complexes with fructopyranose ligands were prepared and investigated for cytotoxicity in the MCF-7 human breast cancer cell line. The antiproliferative effects depended on the presence of isopropylidene protecting groups in the carbohydrate ligand and correlated with the cellular concentration of the complexes. IC(50) values of > 20 microM demonstrated that the fructose derivatives were only moderately active compared to the references auranofin and the aspirin (ASS) derivative [2-acetoxy(2-propynyl)benzoate]hexacarbonyldicobalt (Co-ASS). In continuation of our studies on the mode of action of cobalt-alkyne complexes we studied the influence of the compounds on the formation of 12-HHT (COX-1 product) and 12-HETE (12-LOX product) by human platelets as an indication of the interference in the eicosanoid metabolism, which is discussed as a target system of cytostatics. Co-ASS was an efficient COX-1 inhibitor without LOX inhibitory activity and auranofin inhibited both COX-1 and 12-LOX eicosanoid production. The missing activity of the fructopyranose complexes at the 12-LOX and the only moderate effects at COX-1 indicate that COX/LOX inhibition may be in part responsible for the pharmacological effects of auranofin and Co-ASS but not for those of the fructopyranose complexes.     

Regioselective Hydroxylation of C12–C15 Fatty Acids with Fluorinated Substituents by Cytochrome P450 BM3

We demonstrate herein that wild‐type cytochrome P450 BM3 can recognize non‐natural substrates, such as fluorinated C₁₂–C₁₅ chain‐length fatty acids, and show better catalysis for their efficient conversion. Although the binding affinities for fluorinated substrates in the P450 BM3 pocket are marginally lower than those for non‐fluorinated substrates, spin‐shift measurements suggest that fluoro substituents at the ω‐position can facilitate rearrangement of the dynamic structure of the bulk‐water network within the hydrophobic pocket through a micro desolvation process to expel the water ligand of the heme iron that is present in the resting state. A lowering of the Michaelis–Menten constant (K_m), however, indicates that fluorinated fatty acids are indeed better substrates compared with their non‐fluorinated counterparts. An enhancement of the turnover frequencies (k_cat) for electron transfer from NADPH to the heme iron and for C? H bond oxidation by compound I (Cpd I) to yield the product suggests that the activation energies associated with going from the enzyme–substrate (ES state) to the corresponding transition state (ES^≠ state) are significantly lowered for both steps in the case of the fluorinated substrates. Delicate control of the regioselectivity by the fluorinated terminal methyl groups of the C₁₂–C₁₅ fatty acids has been noted. Despite the fact that residues Arg47/Tyr51/Ser72 exert significant control over the hydroxylation of the subterminal carbon atoms toward the hydrocarbon tail, the fluorine substituent(s) at the ω‐position affects the regioselective hydroxylation. For substrate hydroxylation, we have found that fluorinated lauric acids probably give a better structural fit for the heme pocket than fluorinated pentadecanoic acid, even though pentadecanoic acid is by far the best substrate among the reported fatty acids. Interestingly, 12‐fluorododecanoic acid, with only one fluorine atom at the terminal methyl group, exhibits a comparable turnover frequency to that of pentadecanoic acid. Thus, fluorination of the terminal methyl group introduces additional interactions of the substrate within the hydrophobic pocket, which influence the electron transfers for both dioxygen activation and the controlled oxidation of aliphatics mediated by high‐valent oxoferryl species.     

An unusual isotope effect on substrate inhibition in the oxidation of arachidonic acid by lipoxygenase

Soybean lipoxygenase catalyzes the oxidation of arachidonic acid to 15S-HPETE. The reaction displays strong substrate inhibition with unlabeled substrate but no discernible substrate inhibition with arachidonic acid labeled with deuterium at C13, the site of hydrogen/deuterium atom abstraction. The unusual behavior is due primarily to a large kinetic isotope effect on Km,O2 as a result of the strong selection against deuterium in the abstraction step.     

Structure and catalytic properties of carboxylesterase isozymes involved in metabolic activation of prodrugs

Mammalian carboxylesterases (CESs) comprise a multigene family whose gene products play important roles in biotransformation of ester- or amide-type prodrugs. They are members of an alpha,beta-hydrolase-fold family and are found in various mammals. It has been suggested that CESs can be classified into five major groups denominated CES1-CES5, according to the homology of the amino acid sequence, and the majority of CESs that have been identified belong to the CES1 or CES2 family. The substrate specificities of CES1 and CES2 are significantly different. The CES1 isozyme mainly hydrolyzes a substrate with as mall alcohol group and large acyl group, but its wide active pocket sometimes allows it to act on structurally distinct compounds of either a large or small alcohol moiety. In contrast, the CES2 isozyme recognizes a substrate with a large alcohol group and small acyl group, and its substrate specificity may be restricted by the capability of acyl-enzyme conjugate formation due to the presence of conformational interference in the active pocket. Since pharmacokinetic and pharmacological data for prodrugs obtained from preclinical experiments using various animals are generally used as references for human studies, it is important to clarify the biochemical properties of CES isozymes. Further experimentation for an understanding of detailed substrate specificity of prodrugs for CES isozymes and its hydrolysates will help us to design the ideal prodrugs.     

Electron donor solvent effects provide biosensing with quantum dots

A palmitate biosensor that uses the emission intensity of a semiconducting nanoparticle to report palmitate concentration is presented. This method uses electron transfer to quench the emission from a ZnS-coated CdSe nanoparticle. The fatty acid binding pocket of intestinal fatty acid binding protein is used to modulate the electron transfer properties of [Ru(L)(NH3)4](PF6)2 (L = 5-maleimido-1,10-phenanthroline) that is covalently attached within this pocket. Once the metal-complex-modified protein is attached to ZnS-coated CdSe nanoparticles, palmitate addition excludes water from around the metal complex and increases the electron transfer from the metal complex to the valence band hole of the nanoparticle excited state. A 1.6-fold change in emission intensity is observed upon adding a saturated amount (500 nM) of sodium palmitate. The dissociation constant was calculated as 5 nM with a 1 nM lower limit of detection. Since palmitate does not alter the global conformation of intestinal fatty acid binding protein, palmitate-mediated changes in pocket solvation are suggested. This represents a new method in biosensor construction with semiconducting nanoparticles. Including previous conformation-dependent biosensors, there are thousands of potential analytes that can be detected with these strategies. Such biosensors will provide fluorescence contrast imaging reagents for small molecule analytes.     

The specificity-conferring code of adenylation domains in nonribosomal peptide synthetases.

BACKGROUND: Many pharmacologically important peptides are synthesized nonribosomally by multimodular peptide synthetases (NRPSs). These enzyme templates consist of iterated modules that, in their number and organization, determine the primary structure of the corresponding peptide products. At the core of each module is an adenylation domain that recognizes the cognate substrate and activates it as its aminoacyl adenylate. Recently, the crystal structure of the phenylalanine-activating adenylation domain PheA was solved with phenylalanine and AMP, illustrating the structural basis for substrate recognition. RESULTS: By comparing the residues that line the phenylalanine-binding pocket in PheA with the corresponding moieties in other adenylation domains, general rules for deducing substrate specificity were developed. We tested these in silico 'rules' by mutating specificity-conferring residues within PheA. The substrate specificity of most mutants was altered or relaxed. Generalization of the selectivity determinants also allowed the targeted specificity switch of an aspartate-activating adenylation domain, the crystal structure of which has not yet been solved, by introducing a single mutation. CONCLUSIONS: In silico studies and structure-function mutagenesis have defined general rules for the structural basis of substrate recognition in adenylation domains of NRPSs. These rules can be used to rationally alter the specificity of adenylation domains and to predict from the primary sequence the specificity of biochemically uncharacterized adenylation domains. Such efforts could enhance the structural diversity of peptide antibiotics such as penicillins, cyclosporins and vancomycins by allowing synthesis of 'unnatural' natural products.