Variation In Substrate Structure Research Articles

Morphological differentiation in African weakly electric fish (genus Campylomormyrus) relates to substrate preferences

Under an ecological speciation scenario, the radiation of African weakly electric fish (genus Campylomormyrus) is caused by an adaptation to different food sources, associated with diversification of the electric organ discharge (EOD). This study experimentally investigates a phenotype–environment correlation to further support this scenario. Our behavioural experiments showed that three sympatric Campylomormyrus species with significantly divergent snout morphology differentially react to variation in substrate structure. While the short snout species (C. tamandua) exhibits preference to sandy substrate, the long snout species (C. rhynchophorus) significantly prefers a stone substrate for feeding. A third species with intermediate snout size (C. compressirostris) does not exhibit any substrate preference. This preference is matched with the observation that long-snouted specimens probe deeper into the stone substrate, presumably enabling them to reach prey more distant to the substrate surface. These findings suggest that the diverse feeding apparatus in the genus Campylomormyrus may have evolved in adaptation to specific microhabitats, i.e., substrate structures where these fish forage. Whether the parallel divergence in EOD is functionally related to this adaptation or solely serves as a prezygotic isolation mechanism remains to be elucidated.

Substrate Promiscuity of a Paralytic Shellfish Toxin Amidinotransferase.

Secondary metabolites are assembled by enzymes that often perform reactions with high selectivity and specificity. Many of these enzymes also tolerate variations in substrate structure, exhibiting promiscuity that enables various applications of a given biocatalyst. However, initial enzyme characterization studies frequently do not explore beyond the native substrates. This limited assessment of substrate scope contributes to the difficulty of identifying appropriate enzymes for specific synthetic applications. Here, we report the natural function of cyanobacterial SxtG, an amidinotransferase involved in the biosynthesis of paralytic shellfish toxins, and demonstrate its ability to modify a breadth of non-native substrates. In addition, we report the first X-ray crystal structure of SxtG, which provides rationale for this enzyme's substrate scope. Taken together, these data confirm the function of SxtG and exemplify its potential utility in biocatalytic synthesis.

Study on the mechanism of isomerization of oxaspirohexane catalyzed by Zeise’s Dimer

Mechanism of the rearrangement of oxaspirohexane to 3-methylenetetrahydrofuran under the catalysis of Zeise’s dimer has been studied using DFT methods. Our results reveal that the catalyst, at the initial stage, undergoes a considerable change in structure under the influence of the substrate and the two metal atoms bind the substrate at different positions. Rearrangement of the substrate molecule then takes place to generate the tetrahydrofuran derivative through the formation of platinacyclobutane intermediate, as revealed in the experimental report. Depending on the variation of substrate structure and environmental condition the diversion of the mechanistic pathway to generate different products can also be explained by the DFT methods. Application of several functionals in calculating the global activation barrier also reveals that M06 functional is the most suitable choice to deal with this type of problem and to predict the global activation barrier.

Effects of different substrates on the sprint performance of lizards

The variation in substrate structure is one of the most important determinants of the locomotor abilities of lizards. Lizards are found across a range of habitats, from large rocks to loose sand, each of them with conflicting mechanical demands on locomotion. We examined the relationships among sprint speed, morphology and different types of substrate surfaces in species of lizards that exploit different structural habitats (arboreal, saxicolous, terrestrial and arenicolous) in a phylogenetic context. Our main goals were to assess which processes drive variability in morphology (i.e. phylogeny or adaptation to habitat) in order to understand how substrate structure affects sprint speed in species occupying different habitats and to determine the relationship between morphology and performance. Liolaemini lizards show that most morphological traits are constrained by phylogeny, particularly toe 3, the femur and foot. All ecological groups showed significant differences on rocky surfaces. Surprisingly, no ecological group performed better on the surface resembling its own habitat. Moreover, all groups exhibited significant differences in sprint speed among the three different types of experimental substrates and showed the best performance on sand, with the exception of the arboreal group. Despite the fact that species use different types of habitats, the highly conservative morphology of Liolaemini species and the similar levels of performance on different types of substrates suggest that they confer to the 'jack of all trades and master of none' principle.

The Multiple Substrate Recognition Properties of Ribonuclease P: Achieving Uniformity in Processing Kinetics Despite Variation in Substrate Structure

Ribonuclease P is an essential enzyme that is responsible for catalyzing the maturation of the 5’ end of transfer RNA. In Bacteria, the RNase P holoenzyme is composed of a RNA subunit and a protein subunit. The RNA subunit of RNase P contains the enzyme active site and has the ability to process substrates in the absence of the protein component in vitro, but works as a holoenzyme in vivo. While nucleotide recognition elements adjacent to the site of processing have been identified, different tRNA sequences vary considerably. Remarkably, rates of pre-tRNA processing are uniform despite this variation in substrate sequence and structure. The mechanistic basis for multiple substrate recognition by the holoenzyme is the focus of this study, with the ultimate goal being a better understanding of uniformity in ptRNA processing and discriminates between cognate and non-cognate RNAs. We are determining kinetic schemes for a number of pre-tRNAs using fluorescence assays and standard discontinuous assays. Multiple turnover kinetics have been obtained for ptRNA608MET, a consensus pre-tRNA, and ptRNA605f-MET a non-consensus sequence. Both ptRNAs display a similar Vmax, and Km values. Neither tRNA displayed any burst nor lag phases in pre-steady state kinetics implying that the rate limiting step for our processing model is catalysis. Our initial hypothesis is that uniformity in substrate kcat/Km values results from differential 5’ leader sequence interactions with the protein that compensate for deviations from tRNA consensus recognition sequences. To obtain more insight into sequence and structure influence, we have set up a series of multiple turnover experiments with a consensus and non-consensus ptRNA competing against one another.

Biotransformations with nitrilases

The range of possible nitrilase applications has been recently broadened but in most cases the parameters of the reactions need to be improved to establish viable industrial processes. To achieve this goal, several methods have been used, primarily in screening for enzymes from new sources, enzyme improvement, substrate structure modification, medium engineering, and variation in process parameters. New nitrilases have been obtained by for example genome mining and the metagenomic approach. Protein engineering revealed targets for altering substrate specificity, activity or enantioselectivity, and for changing the acid:amide ratio in the nitrilase product. Variation in substrate structure proved to be another means to modify the enantioselectivity and chemoselectivity of the reactions. The utility of nitrilases will be increased further if they can be applied in combination with other enzymes or chemical catalysts without laborious intermediate isolation. Such processes have been developed, for instance, for the synthesis of (S)-mandelic acid, (S)-mandeloamide or glycolic acid.

Probing Synergy between Two Catalytic Strategies in the Glycoside Hydrolase O-GlcNAcase Using Multiple Linear Free Energy Relationships

Human O-GlcNAcase plays an important role in regulating the post-translational modification of serine and threonine residues with beta-O-linked N-acetylglucosamine monosaccharide unit (O-GlcNAc). The mechanism of O-GlcNAcase involves nucleophilic participation of the 2-acetamido group of the substrate to displace a glycosidically linked leaving group. The tolerance of this enzyme for variation in substrate structure has enabled us to characterize O-GlcNAcase transition states using several series of substrates to generate multiple simultaneous free-energy relationships. Patterns revealing changes in mechanism, transition state, and rate-determining step upon concomitant variation of both nucleophilic strength and leaving group abilities are observed. The observed changes in mechanism reflect the roles played by the enzymic general acid and the catalytic nucleophile. Significantly, these results illustrate how the enzyme synergistically harnesses both modes of catalysis; a feature that eludes many small molecule models of catalysis. These studies also suggest the kinetic significance of an oxocarbenium ion intermediate in the O-GlcNAcase-catalyzed hydrolysis of glucosaminides, probing the limits of what may be learned using nonatomistic investigations of enzymic transition-state structure and offering general insights into how the superfamily of retaining glycoside hydrolases act as efficient catalysts.

Asymmetric transesterification of secondary alcohols catalyzed by feruloyl esterase from Humicola insolens

A new asymmetric transesterification of secondary alcohols catalyzed by feruloyl esterase from Humicola insolens has been found. Although alcohols are not the natural substrates for this enzyme, a high R enantioselectivity was observed. Stereochemical studies showed that variations in substrate structure lead to strong variations in enantioselectivity. The highest enantioselectivities are obtained when the β-carbon of the secondary alcohol is tertiary or quaternary.

Allylic Stereocontrol of the Intramolecular Diels-Alder Reaction

The stereochemical outcome of the intramolecular Diels-Alder reaction of ester-linked 1,3,8-nonatrienes can be controlled by substituents about a stereogenic center attached to C1. The scope and limitations of this approach have been investigated, with variation in substrate structure about the allylic stereocenter and the dienophile. The stereochemical outcomes of these reactions are explained by reference to B3 LYP/6-31G(d) transition structures. New insights into the conformational preferences of allylic alcohol derivatives are reported, results which allow an explanation of the differing levels of pi-diastereofacial selectivity and cis/trans (i.e. endo/exo) selectivity from the reaction.

N-Nitroso-N,O-dialkylhydroxylamines: preparation, structure, and mechanism of the hydronium ion catalysed solvolytic nitrous oxide extrusion reaction

Eleven N-nitroso-N,O-dialkylhydroxylamines, RN(NO)OR′, have been prepared and the mechanisms of their hydronium ion catalysed solvolyses in aqueous solution which liberate nitrous oxide have been investigated. All reactions are first-order in substrate and first-order in hydronium ion, and the second-order rate constants at 25 °C vary over a range of less than 140 in spite of considerable variation in substrate structure (R ranges from methyl to 4-methoxybenzyl to 2-adamantyl, for example) and changes in solvent composition (water with up to 50% methanol or 66% acetonitrile). Enthalpies and entropies of activation are qualitatively similar throughout the range (ΔH‡ = 72–93 kJ mol−1 and ΔS‡ = −19 to −57 J K−1 mol−1) which, with the product analyses, are accommodated by a mechanism involving pre-equilibrium protonation of the substrates followed by rate-limiting dissociation to give RN2O+ and HOR′. The oxodiazonium ion intermediate, RN2O+, then dissociates further to give the carbenium ion intermediate, R+, or suffers direct nucleophilic displacement of N2O by solvent (the external nucleophile) or by R′OH (the internal nucleophile liberated in the initial fragmentation). The carbenium ion, R+ (if formed), suffers nucleophilic capture either by solvent or by R′OH. When acetonitrile is the co-solvent (rather than methanol) for the N-(2-adamantyl) substrate 3g, the product of the Ritter reaction, 2-acetamidoadamantane, is detected. These nitrous oxide liberating reactions are compared with the nitric oxide liberating reactions of related N-nitrosohydroxylamines, and the origin of the difference between them is identified. The N(1)-nitroso group in the N,O-dibenzyl compound 3c is shown by X-ray crystallography to be essentially coplanar with the C and O atoms also bonded to N(1).

Molecular recognition of diketide substrates by a beta-ketoacyl-acyl carrier protein synthase domain within a bimodular polyketide synthase.

Modular polyketide synthases (PKSs) are large multifunctional proteins that catalyze the biosynthesis of structurally complex bioactive products. The modular organization of PKSs has allowed the application of a combinatorial approach to the synthesis of novel polyketides via the manipulation of these biocatalysts at the genetic level. The inherent specificity of PKSs for their natural substrates, however, may place limits on the spectrum of molecular diversity that can be achieved in polyketide products. With the aim of further understanding PKS specificity, as a route to exploiting PKSs in combinatorial synthesis, we chose to examine the substrate specificity of a single intact domain within a bimodular PKS to investigate its capacity to utilize unnatural substrates. We used a blocked mutant of a bimodular PKS in which formation of the triketide product could occur only via uptake and processing of a synthetic diketide intermediate. By introducing systematic changes in the native diketide structure, by means of the synthesis of unnatural diketide analogs, we have shown that the ketosynthase domain of module 2 (KS2 domain) in 6-deoxyerythronolide B synthase (DEBS) tolerates a broad range of variations in substrate structure, but it strongly discriminates against some others. Defining the boundaries of substrate recognition within PKS domains is crucial to the rationally engineered biosynthesis of novel polyketide products, many of which could be prepared only with great difficulty, if at all, by direct chemical synthesis or semi-synthesis. Our results suggest that the KS2 domain of DEBS1 has a relatively relaxed specificity that can be exploited for the design and synthesis of medicinally important polyketide products.

Stereoselectivity of microbial lipases. The substitution at position sn-2 of triacylglycerol analogs influences the stereoselectivity of different microbial lipases.

In the present study, the stereoselectivity of purified lipases from Candida rugosa, Chromobacterium viscosum, Pseudomonas species and Rhizopus arrhizus towards triacylglycerols in comparison to various structural analogs were investigated. Different triacylglycerol analogs with distinct polarities at position sn-2 of the glycerol backbone (1,3-diacyl-2-X-glycerol, where 2-X = 2-acyloxy, 2-alkyloxy, 2-deoxy-2-alkyl, or 2-deoxy-2-phenyl) were synthesized. Substrate hydrophobicity and steric requirement was modified by variation of the alkyl and acyl chain length. Hydrolysis of these substrates demonstrated that minor structural variations at C2 of triacylglycerol strongly affect the stereoselectivity of the lipases tested. It was noteworthy that the variation of substrate structure did not only affect the quantity of stereoselectivity expressed as percentage enantiomeric excess, but also resulted in a reversal of stereopreference in some cases. Replacement of the acylester in position 2 of glycerol by a non-ester-linked aliphatic moiety shifted the preference of Chromobacterium viscosum lipase from sn-3 to sn-1. Lipases from Chromobacterium viscosum. Pseudomonas species and Rhizopus arrhizus exhibited sn-3 preference with 2-deoxy-2-phenyl analogs, while towards substrates with a 2-deoxy-2-alkyl moiety sn-1 stereobias was recorded. Candida rugosa lipase was rather insensitive to substrate variations concerning the polarity at position 2 of the glycerol backbone. However, variation of the acyl chain length significantly influenced stereoselectivity of this lipase.

Maleylacetoacetate cis-trans isomerase: One-step double cis-trans isomerization of monomethyl muconate and the enzyme's probable role in benzene metabolism

Maleylacetoacetate cis-trans isomerase together with glutathione has been found to isomerize cis-trans isomers of monomethyl muconate. Isomerization about a single double bond and concerted double isomerization of the diene unit occurs. In addition to the variations in substrate structure previously identified the current results demonstrate that a cis,cis diene skeleton and a conjugated ester function are accepted by the enzyme. The present work and the fiding of trans,trans-muconic acid in the urine of benzene-fed mice ( M. M. Gad-El-Karim, V. M. Sadagopa Ramanujam, and M. S. Legator (1985) Xenobiotica 15, 211) suggest that maleylacetoacetate cis-trans isomerase may be responsible for the geometrical isomerization. However, cis,cis-muconaldehydic acid rather than cis,cis-muconic acid is suggested to be the early intermediate in benzene metabolism capable of rapid enzyme-catalyzed cis-trans isomerization.

Enzymic synthesis of peptide bonds. IV. Effects of variation in substrate structure on relative extents of synthesis of benzoylamino acid anilides as catalyzed by papain and ficin

Summary From studies with related substrates it has been shown that the previously observed greater reactivity of benzoylleucine than of benzoylvaline, in the papain-catalyzed synthesis of anilides, is in accord with steric hindrance by the branching methyl in the valine substrate. This explanation invokes the principle of steric hindrance in enzyme-substrate interaction involving l -type substrates. The theoretical inferences of this sort of result have been indicated. The relative rankings of 13 benzamino acids in the above reaction were shown to differ when ficin was the catalyst, from the results obtained with papain. The effect of the position of the methyl substituent, however, again dominated in the contrast between leucine and valine. The relationships of yield to dilution of enzyme have been found to vary both with each of the enzymes and with the substrate. Newly obtained anilides have been characterized.