Characterization of the aminocoumarin ligase SimL from the simocyclinone pathway and tandem incubation with NovM,P,N from the novobiocin pathway

Simocyclinone D(8) consists of an anguicycline C-glycoside tethered by a tetraene diester linker to an aminocoumarin. Unlike the antibiotics novobiocin, clorobiocin, and coumermycin A(1), the phenolic hydroxyl group of the aminocoumarin in simocyclinone is not glycosylated with a decorated noviosyl moiety that is the pharmacophore for targeting bacterial DNA gyrase. We have expressed the Streptomyces antibioticus simocyclinone ligase SimL, purified it from Escherichia coli, and established its ATP-dependent amide bond forming activity with a variety of polyenoic acids including retinoic acid and fumagillin. We have then used the last three enzymes from the novobiocin pathway, NovM, NovP, and NovN, to convert a SimL product to a novel novobiocin analogue, in which the 3-prenyl-4-hydroxybenzoate of novobiocin is replaced with a tetraenoate moiety, to evaluate antibacterial activity.     

An unusual amide synthetase (CouL) from the coumermycin A1 biosynthetic gene cluster from Streptomyces rishiriensis DSM 40489.

The aminocoumarin antibiotic coumermycin A1 produced by Streptomyces rishiriensis DSM 40489 contains two amide bonds. The biosynthetic gene cluster of coumermycin contains a putative amide synthetase gene, couL, encoding a protein of 529 amino acids. CouL was overexpressed as hexahistidine fusion protein in Escherichia coli and purified by metal affinity chromatography, resulting in a nearly homogenous protein. CouL catalysed the formation of both amide bonds of coumermycin A1, i.e. between the central 3-methylpyrrole-2,4-dicarboxylic acid and two aminocoumarin moieties. Gel exclusion chromatography showed that the enzyme is active as a monomer. The activity was strictly dependent on the presence of ATP and Mn2+ or Mg2+. The apparent Km values were determined as 26 micro m for the 3-methylpyrrole-2,4-dicarboxylic acid and 44 micro m for the aminocoumarin moiety, respectively. Several analogues of the pyrrole dicarboxylic acid were accepted as substrates. In contrast, pyridine carboxylic acids were not accepted. 3-Dimethylallyl-4-hydroxybenzoic acid, the acyl component in novobiocin biosynthesis, was well accepted, despite its structural difference from the genuine acyl substrate of CouL.     

Cloning and analysis of the simocyclinone biosynthetic gene cluster of <Emphasis Type="Italic">Streptomyces antibioticus</Emphasis> Tü 6040

The biosynthetic gene cluster of the aminocoumarin antibiotic simocyclinone D8 was cloned by screening a cosmid library of Streptomyces antibioticusTü 6040 with a heterologous probe from a gene encoding a cytochrome P450 enzyme involved in the biosynthesis of the aminocoumarin antibiotic novobiocin. Sequence analysis of a 39.4-kb region revealed the presence of 38 ORFs. Six of the identified ORFs showed striking similarity to genes from the biosynthetic gene clusters of the aminocoumarin antibiotics novobiocin and coumermycin A(1). Simocyclinone also contains an angucyclinone moiety, and 12 of the ORFs showed high sequence similarity to biosynthetic genes of other angucyclinone antibiotics. Possible functions within the biosynthesis of simocyclinone D8 could be assigned to 23 ORFs by comparison with sequences in GenBank. Experimental proof for the function of the identified gene cluster was provided by a gene inactivation experiment, which resulted in the abolishment of the formation of the aminocoumarin moiety of simocyclinone. Feeding of the mutant with the aminocoumarin moiety of novobiocin led to a new, artificial simocyclinone derivative.     

Colorimetric inorganic pyrophosphate assay using a double cycling enzymatic method

The biosynthesis of aminocoumarin antibiotics involves the action of amide synthetases which construct amide bonds between aminocoumarins and various acyl moieties. Libraries of aminocoumarin analogues have been generated by in vivo fermentation, via feeding known amide synthetase substrates into producing microbial strains. Critically, such feeding studies rely on the inherent or engineered substrate promiscuity of each amide synthetase. We have initiated a program of directed evolution in order to create mutant amide synthetases for the synthesis of new nonnatural amino coumarin analogues. We used the clorobiocin enzyme CloL as a model amide synthetase to design and validate a fluorimetric high-throughput screen, which can be used to report the activity of mutant amide synthetases toward a broad range of coumarin and acyl donor substrates. Our assay monitors the decrease in fluorescence of aminocoumarins on acylation. The utility of the assay was illustrated by screening a library of amide synthetase mutants created by error-prone PCR. The substrate specificity of an amide synthetase was also rapidly probed using this assay, affording several newly identified substrates. It is anticipated that this high-throughput screen will accelerate the creation of amide synthetase mutants with new specificities by directed evolution.     

Methionine ligation strategy in the biomimetic synthesis of parathyroid hormones

In biological systems, both proteolysis and aminolysis of amide bonds produce activated intermediates through acyl transfer reactions either inter- or intramolecularly. Protein splicing is an illustrative example that proceeds through a series of catalyzed acyl transfer reactions and culminates at an O- or S-acyl intermediate. This intermediate leads to an uncatalyzed acyl migration to form an amide bond in the spliced product. A ligation method mimicking the uncatalyzed final steps in protein splicing has been developed utilizing the acyl transfer amide-bond feature for the blockwise coupling of unprotected, free peptide segments at methionine (Met). The latent thiol moiety of Met can be exploited using homocysteine at the α-amino terminal position of a free peptide for transthioesterification with another free peptide containing an α-thioester to give an S-acyl intermediate. A subsequent, proximity-driven S- to N-acyl migration of this acyl intermediate spontaneously rearranges to form a homocysteinyl amide bond. S-methylation with excess p-nitrobenezensulfonate yields Met at the ligation site. The methionine ligation is selective and orthogonal, and is usually completed within 4 h when performed at slightly basic pH and under strongly reductive conditions. No side reactions due to acylation were observed with any other α-amines of both peptide segments as seen in the synthesis of parathyroid hormone peptides. Furthermore, cyclic peptide can also be obtained through the same strategy by placing both homocysteine at the amino terminus and the thioester at the carboxyl terminus in an unprotected peptide precursor. These biomimetic ligation strategies hold promise for engineering novel peptides and proteins. © 1998 John Wiley & Sons, Inc. Biopoly 46: 319–327, 1998     

Biosynthesis of clorobiocin: investigation of the transfer and methylation of the pyrrolyl-2-carboxyl moiety

Clorobiocin is an aminocoumarin antibiotic containing a 5-methylpyrrolyl-2-carboxyl moiety, attached by an ester bond to a deoxysugar. This pyrrolyl moiety is important for the binding of the antibiotic to its biological target, the B subunit of gyrase. Inactivation experiments had shown that two putative acyl carrier proteins, CloN5 and CloN1, and two putative acyl transferases, CloN2 and CloN7, are involved in the transfer of the pyrrolyl-2-carboxyl moiety to the deoxysugar. In this study, pyrrolyl-2-carboxyl-N-acetylcysteamine thioester was synthesized and fed to cloN1 ⁻ , cloN2 ⁻ and cloN7 ⁻ mutants, and secondary metabolite formation was analyzed by HPLC and HPLC-MS. Transfer of the pyrrolyl-2-carboxyl moiety was observed in the cloN1 ⁻ and cloN2 ⁻ mutants, but not in the cloN7 ⁻ mutant, suggesting that CloN7 is responsible for this reaction. The product of this transfer, novclobiocin 109, was not further methylated to the 5-methylpyrrolyl-2-carboxyl compound, i.e. clorobiocin, suggesting that methylation does not take place after the acyl transfer. Additional investigations for the presence of 5-methylpyrrolyl-2-carboxylic acid in the mutants, and inactivation experiments with the methyltransferase gene cloN6, suggested that methylation by CloN6 and acyl transfer by CloN7 take place in a concerted fashion, requiring the presence of both proteins for efficient product formation. A mechanism for the methylation/acyl transfer process in the late steps of clorobiocin biosynthesis, involving CloN1, CloN2, CloN5, CloN6 and CloN7 is suggested.     

Peptide Thioester Synthesis via an Auxiliary-Mediated N–S Acyl Shift Reaction in Solution

The 4,5-dimethoxy-2-mercaptobenzyl (Dmmb) group attached to a main chain amide in a peptide is easily transformed into an S-peptide via an intramolecular N–S acyl shift reaction under acidic conditions, and the S-peptide produces a peptide thioester through an intermolecular thiol–thioester exchange reaction. In order to develop a method for efficiently preparing peptide thioesters based on the N–S acyl shift reaction, the factors involved in this process were analyzed in detail. The general features of the transformation at the Dmmb group attached amide bond in a trifluoroacetic acid (TFA) solution and the generation of a peptide thioester were examined by ¹³C-NMR spectral measurements, reversed-phase (RP) HPLC analyses, mass measurements, and amino acid analyses. The methoxy group of the Dmmb group was not essential for the N–S acyl shift reaction, but played a role in stabilizing the thioester form. The addition of water to the TFA solution accelerated the N–S acyl shift reaction mediated by the Dmmb group and also suppressed the acid-catalyzed cleavage of the Dmmb group. A peptide thioester was produced from the S-peptide via an intermolecular thiol–thioester exchange reaction with minimal epimerization of the amino acid residue that constituted the thioester bond. Undesirable side reactions, such as the hydrolysis of the thioester bond and an S–N acyl shift reaction occurred during the synthetic process, which is a subject of further investigation.     

Purification,characterization, gene cloning and nucleotide sequencing of D-stereospecific amino acid amidase from soil bacterium: Delftia acidovorans

The D-amino acid amidase-producing bacterium was isolated from soil samples using an enrichment culture technique in medium broth containing D-phenylalanine amide as a sole source of nitrogen. The strain exhibiting the strongest activity was identified as Delftia acidovorans strain 16. This strain produced intracellular D-amino acid amidase constitutively. The enzyme was purified about 380-fold to homogeneity and its molecular mass was estimated to be about 50 kDa, on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was active preferentially toward D-amino acid amides rather than their L-counterparts. It exhibited strong amino acid amidase activity toward aromatic amino acid amides including D-phenylalanine amide, D-tryptophan amide and D-tyrosine amide, yet it was not specifically active toward low-molecular-weight D-amino acid amides such as D-alanine amide, L-alanine amide and L-serine amide. Moreover, it was not specifically active toward oligopeptides. The enzyme showed maximum activity at 40°C and pH 8.5 and appeared to be very stable, with 92.5% remaining activity after the reaction was performed at 45°C for 30 min. However, it was mostly inactivated in the presence of phenylmethanesulfonyl fluoride or Cd²⁺, Ag⁺, Zn²⁺, Hg²⁺ and As³⁺ . The NH₂ terminal and internal amino acid sequences of the enzyme were determined; and the gene was cloned and sequenced. The enzyme gene damA encodes a 466-amino-acid protein (molecular mass 49,860.46 Da); and the deduced amino acid sequence exhibits homology to the D-amino acid amidase from Variovorax paradoxus (67.9% identity), the amidotransferase A subunit from Burkholderia fungorum (50% identity) and other enantioselective amidases.

     

New Ras CAAX mimetics: Design, synthesis, antiproliferative activity, and RAS prenylation inhibition

Mimetics of the C-terminal CAAX tetrapeptide of Ras protein were designed replacing cysteine (C) by 2-hydroxymethylbenzodioxane or 2-aminomethylbenzodioxane, respectively etherified and amidified with 2′-methyl or 2′-methoxy substituted 2-carboxy-4-hydroxybiphenyl and 2,4-dicarboxybiphenyl. These pluri-substituted biphenyl systems, used as internal spacer and AA dipeptide bioisoster, were linked to the methyl ester of l-methionine, glycine or l-leucine by an amide bond. The resultant twelve pairs of stereoisomers at the dioxane C-2 were tested for antiproliferative effect finding the maximum activity for derivatives with methyleneoxy linker between benzodioxane and 2′-methylbiphenyl. Of these compounds, the one with terminal methionine and S configuration proved a good Ras prenylation inhibitor in a cell-based assay.     

Structural Insights into the Acyl Intermediates of the Plasmodium falciparum Fatty Acid Synthesis Pathway: THE MECHANISM OF EXPANSION OF THE ACYL CARRIER PROTEIN CORE*

Acyl carrier protein (ACP) plays a central role in fatty acid biosynthesis. However, the molecular machinery that mediates its function is not yet fully understood. Therefore, structural studies were carried out on the acyl-ACP intermediates of Plasmodium falciparum using NMR as a spectroscopic probe. Chemical shift perturbation studies put forth a new picture of the interaction of ACP molecule with the acyl chain, namely, the hydrophobic core can protect up to 12 carbon units, and additional carbons protrude out from the top of the hydrophobic cavity. The latter hypothesis stems from chemical shift changes observed in C_α and C_β of Ser-37 in tetradecanoyl-ACP. ¹³C,¹⁵N-Double-filtered nuclear Overhauser effect (NOE) spectroscopy experiments further substantiate the concept; in octanoyl (C₈)- and dodecanoyl (C₁₂)-ACP, a long range NOE is observed within the phosphopantetheine arm, suggesting an arch-like conformation. This NOE is nearly invisible in tetradecanoyl (C₁₄)-ACP, indicating a change in conformation of the prosthetic group. Furthermore, the present study provides insights into the molecular mechanism of ACP expansion, as revealed from a unique side chain-to-backbone hydrogen bond between two fairly conserved residues, Ile-55 HN and Glu-48 O. The backbone amide of Ile-55 HN reports a pK_a value for the carboxylate, ∼1.9 pH units higher than model compound value, suggesting strong electrostatic repulsion between helix II and helix III. Charge-charge repulsion between the helices in combination with thrust from inside due to acyl chain would energetically favor the separation of the two helices. Helix III has fewer structural restraints and, hence, undergoes major conformational change without altering the overall-fold of P. falciparum ACP.In the malarial parasite Plasmodium falciparum, fatty acid biosynthesis occurs by a pathway distinct from the host. A number of enzymes involved in the process viz. β-ketoacyl acyl carrier protein (ACP)⁵ synthase III, β-hydroxy acyl-ACP hydratase, and enoyl-ACP reductase are targets for drug design (1, 2). An indispensable component, crucial for each step of the pathway, is a small acidic protein, the ACP. ACP plays a pivotal role in a range of biochemical processes, like fatty acid biosynthesis (3), polyketide synthesis (4, 5), oligosaccharides (6), biotin, and nonribosomal peptide synthesis (7, 8). Thus, the knowledge of structural features, which dictate ACP function, could offer new avenues for inhibitor design to disable several pathways of the parasite in parallel.Acyl carrier protein differs structurally in the host and the parasite. It exists as an independent protein in type II fatty acid synthesis pathway, observed in P. falciparum, Escherichia coli, spinach, and most prokaryotes. In the type II pathway, fatty acids are synthesized by multiple enzymes catalyzing different reactions. Conversely, mammalian ACP (malarial host) is an integral domain of one single multidomain, multifunctional fatty acid synthase (FAS) (type I pathway), each domain catalyzing a particular reaction. Interestingly, ACPs of type I and II pathway share a similar fold, the ACP molecule of type II pathway can be substituted with the ACP domain of type I pathway in some cases, and the latter is recognized as a substrate in vitro by key enzymes of type II pathway (9).The primary function of ACP is to shuttle the lengthening acyl chains to the catalytic site of FAS enzymes. It is expressed as an apoprotein (inactive) and modified to holo-ACP (active) by the transfer of a 4′-phosphopantetheine moiety from coenzyme A (CoA) to a conserved serine residue, Ser-36/37, with ACP synthase acting as a catalyst. The acyl chain gets covalently tethered to the terminal cysteamine thiol of the 4′-phosphopantetheine prosthetic group, which in turn transfers the acyl chain to the respective enzymes during elongation. Biosynthesis of fatty acid(s) is initiated by the carboxylation of acetyl-CoA to malonyl-CoA, which is transacylated to malonyl-ACP. Malonyl-ACP condenses with acetyl-CoA, resulting in the formation of enoyl-/butyryl-ACP (C₄-) which enters the elongation cycle. Two carbon atoms are added per elongation cycle, resulting in acyl-ACPs C₆-, C₈-, C₁₀-, C₁₂-, C₁₄-, and C₁₆-ACP. Palmitate (C₁₆) is the most common product of type I pathway, whereas in the type II pathway, products range from saturated to unsaturated, branched, unbranched, or variable chain lengths.Structurally, ACP is a four-helix bundle protein, with the helices enclosing a central hydrophobic cavity (10–17). In the type II pathway, the hydrophobic cavity accommodates the growing acyl chain and the β-mercaptoethyl moiety of the 4′-phosphopantetheine arm. The acyl chain remains embedded in the cavity, which expands with increasing length of the acyl chain as observed in E. coli and spinach (12). The mechanism of acyl chain interaction with the ACP molecule is remarkably different in rat, which belongs to the type I fatty acid pathway. Insignificant interactions between the ACP molecule and the acyl chain are observed, suggesting that the ACP molecule does not sequester the acyl chain, and therefore, the acyl chain in type I pathway is protected in a way different from the type II pathway (18).Despite the availability of structural data for a number of acyl-ACPs e.g. E. coli and spinach (12, 14, 19, 20), molecular details pertaining to acyl chain carriage and its presentation to the FAS enzymes of type II pathway is still an enigma. The general consensus is that the ACP molecule can accommodate 10 carbon atoms only. In spinach, the hydrophobic cavity of ACP expands to accommodate acyl chain lengths ranging from C_10:0 to C_18:0. However, chains longer than 10 carbon units are not fully protected (14). In E. coli, 10 carbon atoms have been observed to be accommodated in the hydrophobic core (19). A molecular dynamics study on E. coli published recently also shows that the hydrophobic core of ACP can hold a maximum of 10 carbon atoms only (21). Here, we demonstrate that P. falciparum ACP (PfACP) can protect more than 10-carbon-atom-long acyl chains, with a maximum of 12 carbon atoms. An in silico study on PfACP published recently proposes the possible mechanism of substrate delivery based on steered molecular dynamics simulations using E. coli acyl-ACPs as the starting model (22). There are no experimental data (x-ray or NMR) available to date on the acyl-ACPs of P. falciparum. Present work for the first time provides structural insights into the acyl-PfACP intermediates using NMR as a primary tool. The precision and sensitivity of NMR allowed identification of key interactions between the acyl chain and the ACP molecule, leading to the proposal of a model unraveling the sequence of structural changes accompanying acyl chain insertion. The molecular basis of ACP expansion in PfACP upon acyl chain elongation has also been deciphered.     

A rapid posttranslational myristylation of a 68-kD protein in D. discoideum

Cells incubated with [3H]myristate were shown to rapidly and specifically acylate a 68-kD protein, p68, in a developmentally-regulated manner. The fatty acid incorporated into p68 was identified as myristate, and is linked to the protein via an amide bond, apparently to an NH2-terminal glycine. The acylation of p68 in D. discoideum displays some unusual properties. Unexpectedly, myristylation of p68 is a posttranslational event and occurs in the presence of inhibitors of protein synthesis. Another unusual finding was that although p68 is a stable protein, the acyl moiety is removed with a half time of approximately 15 min.     

A comparative study of the backbone dynamics of two closely related lipid binding proteins: Bovine heart fatty acid binding protein and porcine ileal lipid binding protein

The backbone dynamics of bovine heart fatty acid binding protein (H-FABP) and porcine ileal lipid binding protein (ILBP) were studied by 15N NMR relaxation (T1 and T2) and steady state heteronuclear 15N{1H} NOE measurements. The microdynamic parameters characterizing the backbone mobility were determined using the model-free approach. For H-FABP, the non-terminal backbone amide groups display a rather compact protein structure of low flexibility. In contrast, for ILBP an increased number of backbone amide groups display unusually high internal mobility. Furthermore, the data indicate a higher degree of conformational exchange processes in the sec-msec time range for ILBP compared to H-FABP. These results suggest significant differences in the conformational stability for these two structurally highly homologous members of the fatty acid binding protein family.     

The effect of 17O on the relaxation of an amide proton within a hydrogen bond

Summary The relaxation rates of the multiple-quantum coherence for the amide hydrogen of Gly¹³ in ras p21·GDP were determined in the presence and absence of ¹⁷O labeling in the -phosphate of GDP. No significant difference could be observed between labeled and unlabeled samples, in spite of the fact that the hydrogen bond from the amide group of Gly¹³ to the -phosphate is shorter than is typical, based on its chemical shift. For macromolecules in which an oxygen atom is the acceptor of a hydrogen bond, dipolar coupling between ¹⁷O and hydrogen is unlikely to be observable, except for extremely short H-bonds.Abbreviations p21 21-kD protein product of the human H-ras gene - GMPPCP guanylyl [,-methylene]diphosphate - HPLC high-performance liquid chromatography - CSA chemical shift anisotropy - HMQC heteronuclear multiple-quantum coherence     

A switchable stapled peptide

The O‐N acyl transfer reaction has gained significant popularity in peptide and medicinal chemistry. This reaction has been successfully applied to the synthesis of difficult sequence‐containing peptides, cyclic peptides, epimerization‐free fragment coupling and more recently, to switchable peptide polymers. Herein, we describe a related strategy to facilitate the synthesis and purification of a hydrophobic stapled peptide. The staple consists of a serine linked through an amide bond formed from its carboxylic acid function and the side chain amino group of diaminopropionic acid and through an ester bond formed from its amino group and the side chain carboxylic acid function of aspartic acid. The α‐amino group of serine was protonated during purification. Interestingly, when the peptide was placed at physiological pH, the free amino group initiated the O‐N shift reducing the staple length by one atom, leading to a more hydrophobic stapled peptide. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

A new cyclolinopeptide containing nonproteinaceous amino acid N-methyl-4-aminoproline

Summary We have found that besides the known cyclolinopeptides A (CLA) and B (CLB), there is a new cyclic peptide in linseed mill cake that we have named CLX. Its composition is very similar to that of CLA, a cyclic peptide with a distinct immunosuppressive activity. The sequence of this peptide has been established ascyclo(PPFFILLX), where X is a non-proteinaceous amino acid,N-methyl-4-aminoproline. this amino acid substitutes for two amino acid residues of CLA, mimicking a dipeptide moiety with a nonplanarcis amide bond. The non-proteinaceous amino acid X may mimic a transition state of the peptide bond which exists in such processes as, e.g., PPIase-catalysedcis/trans amide-Pro bond isomerisation.     

Caffeic acid phenethyl ester is a potent inhibitor of HIF prolyl hydroxylase: structural analysis and pharmacological implication

Caffeic acid phenethyl ester (CAPE) is an active component of propolis from honeybee. We investigated a potential molecular mechanism underlying a CAPE-mediated protective effect against ischemia/reperfusion (I/R) injury and analyzed the structure contributing to the CAPE effect. CAPE induced hypoxia-inducible factor-1 (HIF-1) α protein, concomitantly transactivating the HIF-1 target genes vascular endothelial growth factor and heme oxygenase-1, which play a protective role in I/R injury. CAPE delayed the degradation of HIF-1α protein in cells, which occurred by inhibition of HIF prolyl hydroxylase (HPH), the key enzyme for von Hippel–Lindau-dependent HIF-1α degradation. CAPE inhibition of HPH and induction of HIF-1α protein were neutralized by an elevated dose of iron. The catechol moiety, a chelating group, is essential for HPH inhibition, while hydrogenation of the double bond (–CC–) in the Michael reaction acceptor markedly reduced potency. Removal of the phenethyl moiety of CAPE (substitution with the methyl moiety) severely deteriorated its inhibitory activity for HPH. Our data suggest that a beneficial effect of CAPE on I/R injury may be ascribed to the activation of HIF-1 pathway via inhibition of HPH and reveal that the chelating moiety of CAPE acted as a pharmacophore while the double bond and phenethyl moiety assisted in inhibiting HPH.     

Enzymatic peptide synthesis by the recombinant proline-specific endopeptidase from Flavobacterium meningosepticum and its mutationally altered Cys-556 variant

Proline-specific endopeptidase (PSE) (EC 3.4.21.26) was investigated for its potential as a catalyst in peptide synthesis. Using an activated peptide ester or a peptide amide as the acyl component, the enzyme catalyzed kinetically controlled aminolysis and transpeptidation respectively, with various amino acid amides as acyl acceptors. To a certain extent the nucleophile preference reflected the amino acid preference in the S₁-position of the enzyme in peptide hydrolysis: the highest fractions of aminolysis were obtained using amino acid amides with hydrophobic side-chains (e.g. Leu-NH₂, Phe-NH₂). PSE also catalyzed the thermodynamically controlled condensation of short peptides with a free carboxyterminus and various amino acid amides. This enabled us to examine the acceptance of different acyl components in the substrate-binding site of the enzyme with regard to their amino acid composition: In the S₁ position proline was clearly favored, but alanine was also accepted, whereas the S₂ subsite accepted various amino acids rather unspecifically. Since PSE was shown to be extremely sensitive against water-miscible organic solvents, an alternative approach was used to increase yields in enzymatic peptide synthesis: a derivative of PSE in which the catalytic Ser-556 is converted to a Cys was constructed by protein engineering. This mutant (PSE_cys) exhibited a dramatically increased peptide ligase activity in aqueous solution.     

Auxin activity and molecular structure of 2-alkylindole-3-acetic acids

2-Methylindole-3-acetic acid (2-Me-IAA) is a known auxin, but its 2-ethyl homologue has been considered inactive. Here we show that the compound previously bioassayed as 2-ethylindole-3-acetic acid (2-Et-IAA) was, in fact, 3-(3-methylindol-2-yl)propionic acid. The proper 2-Et-IAA and its 2-(n-propyl) homologue (2-Pr-IAA) are prepared, unambiguously characterized, and their auxin activity is demonstrated in the Avena coleoptile-section straight-growth test. Their half-optimal concentrations are approximately the same as for 2-Me-IAA (2 × 10^–5mol L^–1), and hence about ten times larger than for unsubstituted indole-3-acetic acid (IAA) and its derivatives alkylated in positions 4, 5, 6 or 7. The optimal response elicited by 2-Et-IAA and 2-Pr-IAA is about half that observed for 2-Me-IAA. These characteristics place the three 2-alkyl-IAAs along the borderline between the classes of strong and weak auxins, thus corroborating the results of interaction similarity analysis, a mathematical approach based on the capability of auxin molecules to participate in non-bonding interactions with a generalized receptor protein. X-ray diffraction analysis shows no explicit structural features to be blamed for the decrease in auxin activity caused by attaching a 2-alkyl substituent to the IAA molecule; sterical interference of the 3-CH₂COOH group and the 2-alkyl moiety is barely recognizable in the crystalline state. Quantum-chemical calculations and molecular dynamics simulations suggest that 2-alkyl-IAAs, in the absence of crystal-packing restraints, prefer conformations with the CH₂-COOH bond tilted to the heterocyclic ring system. Substantially higher conformational energy (and hence lower abundance) is predicted for planar conformers which were previously shown to prevail for IAA and many of its derivatives substituted in the benzene moiety of the indole nucleus. This shift in the rotational preferences of the -CH₂COOH moiety may be one of the reasons for the reduced plant-growth promoting activity of 2-alkyl-IAAs.