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New structural motif for carboxylic acid perhydrolases.

ABSTRACT: Some serine hydrolases also catalyze a promiscuous reaction--reversible perhydrolysis of carboxylic acids to make peroxycarboxylic acids. Five X-ray crystal structures of these carboxylic acid perhydrolases show a proline in the oxyanion loop. Here, we test whether this proline is essential for high perhydrolysis activity using Pseudomonas fluorescens esterase (PFE). The L29P variant of this esterase catalyzes perhydrolysis 43-fold faster (k(cat) comparison) than the wild type. Surprisingly, saturation mutagenesis at the 29 position of PFE identified six other amino acid substitutions that increase perhydrolysis of acetic acid at least fourfold over the wild type. The best variant, L29I PFE, catalyzed perhydrolysis 83-times faster (k(cat) comparison) than wild-type PFE and twice as fast as L29P PFE. Despite the different amino acid in the oxyanion loop, L29I PFE shows a similar selectivity for hydrogen peroxide over water as L29P PFE (?(0)=170 vs. 160 M(-1)), and a similar fast formation of acetyl-enzyme (140 vs. 62 U mg(-1)). X-ray crystal structures of L29I PFE with and without bound acetate show an unusual mixture of two different oxyanion loop conformations. The type II ?-turn conformation resembles the wild-type structure and is unlikely to increase perhydrolysis, but the type I ?-turn conformation creates a binding site for a second acetate. Modeling suggests that a previously proposed mechanism for L29P PFE can be extended to include L29I PFE, so that an acetate accepts a hydrogen bond to promote faster formation of the acetyl-enzyme.

SUBMITTER: Yin DT

PROVIDER: S-EPMC3784613 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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New structural motif for carboxylic acid perhydrolases.

Yin DeLu Tyler DT Purpero Vince M VM Fujii Ryota R Jing Qing Q Kazlauskas Romas J RJ

Chemistry (Weinheim an der Bergstrasse, Germany) 20130116 9

Some serine hydrolases also catalyze a promiscuous reaction--reversible perhydrolysis of carboxylic acids to make peroxycarboxylic acids. Five X-ray crystal structures of these carboxylic acid perhydrolases show a proline in the oxyanion loop. Here, we test whether this proline is essential for high perhydrolysis activity using Pseudomonas fluorescens esterase (PFE). The L29P variant of this esterase catalyzes perhydrolysis 43-fold faster (k(cat) comparison) than the wild type. Surprisingly, sat ...[more]

PMID: 23325572

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Project description:BACKGROUND:Co-crystal is a structurally homogeneous crystalline material that contains two or more neutral building blocks that are present in definite stoichiometric amounts. The main advantage of co-crystals is their ability to generate a variety of solid forms of a drug that have distinct physicochemical properties from the solid co-crystal components. In the present investigation, five co-crystals containing 2-amino-6-chloropyridine (AMPY) moiety were synthesized and characterized. RESULTS:The crystal structure of 2-amino-6-chloropyridine (AMPY) (I), and the robustness of pyridine-acid supramolecular synthon were discussed in four stoichiometry co-crystals of AMPY…BA (II), AMPY…2ABA (III), AMPY…3CLBA (IV) and AMPY…4NBA (V). The abbreviated designations used are benzoic acid (BA), 2-aminobenzoic acid (2ABA), 3-chlorobenzoic acid (3CLBA) and 4-nitrobenzoic acid (4NBA). All the crystalline materials have been characterized by (1)HNMR, (13)CNMR, IR, photoluminescence, TEM analysis and X-ray diffraction. The supramolecular assembly of each co-crystal is analyzed and discussed. CONCLUSIONS:Extensive N---H?·?·?·?N/N---H?·?·?·?O/O---H?·?·?·?N hydrogen bonds are found in (I-V), featuring different supramolecular synthons. In the crystal structure, for compound (I), the 2-amino-6-chloropyridine molecules are linked together into centrosymmetric dimers by hydrogen bonds to form homosynthon, whereas for compounds (II-V), the carboxylic group of the respective acids (benzoic acid, 2-aminobenzoic acid, 3-chlorobenzoic acid and 4-nitrobenzoic acid) interacts with pyridine molecule in a linear fashion through a pair of N---H?·?·?·?O and O---H?·?·?·?N hydrogen bonds, generating cyclic hydrogen-bonded motifs with the graph-set notation [Formula: see text] , to form heterosynthon. In compound (II), another intermolecular N---H?·?·?·?O hydrogen bonds further link these heterosynthons into zig-zag chains. Whereas in compounds (IV) and (V), these heterosynthons are centrosymmetrically paired via N---H?·?·?·?O hydrogen bonds and each forms a complementary DADA [D?=?donor and A?=?acceptor] array of quadruple hydrogen bonds, with graph-set notation [Formula: see text], [Formula: see text] and [Formula: see text].

Project description:Using microarray technology and a set of chickpea (Cicer arietinum L.) unigenes and grasspea (Lathyrus sativus L.) ESTs, chickpea responses to treatments with the defence signalling compounds salicylic acid (SA), methyl jasmonate (MeJA), and aminocyclopropane carboxylic acid (ACC) were studied in four chickpea genotypes with ranging levels of resistance to ascochyta blight (Ascochyta rabiei (Pass.) L.). The experimental system minimized environmental effects and was conducted in reference design, where samples from untreated controls acted as references against post-treatment samples. Robust data quality was achieved through the use of three biological replicates (including a dye-swap), the inclusion of negative controls, and strict selection criteria for differentially expressed genes including a fold change cut-off determined by self-to-self hybridizations, Students t test and multiple testing correction (P<0.05). Microarray observations were also validated by quantitative RT-PCR. The time-course expression patterns of 715 experimental microarray features resulted in differential expression of 425 genes in at least one condition. The A. rabiei resistant chickpea genotypes showed a more substantial range of defence-related gene induction by all treatments, indicating that they may possess stronger abilities to resist infection. Further, the involvement of SA, MeJA, and ACC signalling was identified for the regulation of some important A. rabiei responsive genes, as well as cross-talk between these pathways. This study also found evidence to suggest the involvement of A. rabiei-specific signalling mechanisms for the induction of several genes that were previously implicated in A. rabiei resistance. Overall, this study characterised the regulatory mechanisms of many chickpea genes that may be important in defence against various pathogens, as well as other cellular functions. Although the size of the microarray was limited, the results provided novel insights to the molecular control of chickpea cellular processes, which may assist the understanding of chickpea defence mechanisms and allow enhanced development of disease resistant cultivars. Keywords: time course defence-signalling teatment analysis

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New structural motif for carboxylic acid perhydrolases.

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New structural motif for carboxylic acid perhydrolases.

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