Rational redesign of the 4-chlorobenzoate binding site of 4-chlorobenzoate: coenzyme a ligase for expanded substrate range

Environmental aromatic acids are transformed to chemical energy in bacteria that possess the requisite secondary pathways. Some of these pathways rely on the activation of the aromatic acid by coenzyme A (CoA) thioesterification catalyzed by an aromatic acid: CoA ligase. Adaptation of such pathways to the bioremediation of man-made pollutants such as polychlorinated biphenyl (PCB) and dichlorodiphenyltrichloroethane (DDT) requires that the chlorinated benzoic acid byproduct that is formed be able to be eliminated by further degradation. To take advantage of natural benzoic acid degrading pathways requiring initial ring activation by thioesterification, the pathway aromatic acid:CoA ligase must be an effective catalyst with the chlorinated benzoic acid. This study, which focuses on the 4-chlorobenzoate:CoA ligase (CBL) of the 4-monochlorobiphenyl degrading bacterium Alcaligenes sp. strain ALP83, was carried out to determine if the 4-chlorobenzoate binding site of this enzyme can be transformed by rational design to recognize the chlorobenzoic acids formed in the course of breakdown of other environmental PCB congeners. The fundamental question addressed in this study is whether it is possible to add or subtract space from the substrate-binding pocket of this ligase (to complement the topology of the unnatural aromatic substrate) without causing disruption of the ligase catalytic machinery. Herein, we report the results of a substrate specificity analysis that, when interpreted within the context of the X-ray crystal structures, set the stage for the rational design of the ligase for thioesterification of two PCB-derived chlorobenzoic acids. The ligase was first optimized to catalyze CoA thioesterification of 3,4-dichlorobenzoic acid, a poor substrate, by truncating Ile303, a large hydrophobic residue that packs against the ring meta-C(H) group. The structural basis for the approximately 100-fold enhancement in the rate of 3,4-dichlorobenzoate thioesterification catalyzed by the I303A and I303G CBL mutants was validated by determination of the crystal structure of the 3,4-dichlorobenzoate-bound enzymes. Determinations of the structures of I303 mutant complexes of 3-chlorobenzoate, a very poor substrate, revealed nonproductive binding as a result of the inability of the substrate ring C(4)H group to fill the pocket that binds the C(4)Cl group of the native substrate. The C(4)Cl pocket of the CBL I303A mutant was then reduced in size by strategic amino acid replacement. A 54-fold improvement in catalytic efficiency was observed for the CBL F184W/I303A/V209T triple mutant. The results of this investigation are interpreted as evidence that the plasticity of the ligase catalytic scaffold is sufficient to allow expansion of substrate range by rational design. The combination of structural and kinetic analyses of the constructed mutants proved to be an effective approach to engineering the ligase for novel substrates.     

Cloning and characterization of an AtNHX2-like Na+/H+ antiporter gene from Ammopiptanthus mongolicus (Leguminosae) and its ectopic expression enhanced drought and salt tolerance in Arabidopsis thaliana

An orthologue of the vacuolar Na⁺/H⁺ antiporter gene, AmNHX2, was isolated from a desert plant, Ammopiptanthus mongolicus, by RACE-PCR. It has a total length of 1,986 bp, with an open reading frame of 1,632 bp, encoding a predicted polypeptide of 543 amino acids. Sequence similarity and exon constituent analysis clearly suggested that AmNHX2 encoded an AtNHX2 (an antiporter from Arabidopsis) like vacuolar Na⁺/H⁺ antiporter. AmNHX2 could be strongly induced by both drought and salt stress. Heterologous expression in the yeast mutant nhx1 indicated that AmNHX2 was the orthologue of ScNHX1, and the complementation effect was almost the same as AtNHX1. Over-expressing AmNHX2 resulted in enhanced tolerances to both drought and salt stresses in transgenic Arabidopsis plants. The transgenic plants accumulated lower Na⁺ content in their leaves, showing healthier root system after salt stress, and retained more water during the drought stress. Our work suggested that AmNHX2 was a salt tolerance determinant in A. mongolicus, but might not be a contributor to the difference in salt sensitivity between A. thaliana and A. mongolicus.     

Production of a thermostable metal-tolerant laccase from Trametes versicolor and its application in dye decolorization

Production of laccase using a submerged culture of Trametes versicolor sdu-4 was optimized using a central composite design of the Response Surface Methodology. Optimized conditions gave a laccase yield of 4,213 U/L which was approximately three times of that in basal medium. The laccase was purified to homogeneity using a three-step process. The overall yield of the purification was 58%, with a purification fold of 11.4 and a specific activity of 1320.7 U/mg protein. The molecular mass of the laccase was 60 kDa. The optimum pH values of the enzyme were 2.2, 3.7, and 7 for the oxidations of ABTS, DMP, and syringaldazine, respectively. The enzyme had adaptability to a broad pH range and high temperature and wsa stable at pH 3.0 ∼ 10.0. The half-life of this laccase at 70°C was 2.2 h. Methyl red, 2-bromophenol, and 4-bromophenol were oxidized by the purified laccase in the absence of mediators. Purified laccase was effective in the decolorization of several dyes and was not inhibited by Cu²⁺, Mn²⁺, Zn²⁺, Na⁺, K⁺, Mg²⁺, Ba²⁺, and Ca²⁺ at 5 mM. These excellent characteristics made it a highly attractive candidate for industrial use.     

SPOCK1 promotes the invasion and metastasis of gastric cancer through Slug‐induced epithelial‐mesenchymal transition

Metastasis is a crucial impediment to the successful treatment for gastric cancer. SPOCK1 has been demonstrated to facilitate cancer metastasis in certain types of cancers; however, the role of SPOCK1 in the invasion and metastasis of gastric cancer remains elusive. SPOCK1 and epithelial‐mesenchymal transition (EMT)‐related biomarkers were detected by immunohistochemistry and Western blot in gastric cancer specimens. Other methods including stably transfected against SPOCK1 into gastric cancer cells, Western blot, migration and invasion assays in vitro and metastasis assay in vivo were also performed. The elevated expression of SPOCK1 correlates with EMT‐related markers in human gastric cancer tissue, clinical metastasis and a poor prognosis in patients with gastric cancer. In addition, knockdown of SPOCK1 expression significantly inhibits the invasion and metastasis of gastric cancer cells in vitro and in vivo, inversely, SPOCK1 overexpression results in the opposite effect. Interestingly, SPOCK1 expression has no effect on cell proliferation in vitro and in vivo. Regarding the mechanism(s) of SPOCK1‐induced cells invasion and metastasis, we prove that Slug‐induced EMT is involved in SPOCK1‐facilitating gastric cancer cells invasion and metastasis. The elevated SPOCK1 expression is closely correlated with cancer metastasis and patient survival, and SPOCK1 promotes the invasion and metastasis of gastric cancer through Slug‐mediated EMT, thereby possibly providing a novel therapeutic target for gastric cancer.     

Cloning and expression analysis of a water stress-induced gene from Brassica oleracea.

Plant growth and productivity are greatly affected by water stress, such as drought and salinity. Here we report on the cloning and expression analysis of a water stress-induced gene from Brassica oleracea (designated as BoWS, GenBank accession number AY571333) by rapid amplification of cDNA ends (RACE). The full-length cDNA of BoWS consisted of 594 bp and contained a 285 bp open reading frame (ORF) encoding a 95-amino-acid protein. The deduced protein had a calculated molecular mass of 10.53 kDa and an isoelectric point of 6.93. The sequence similarity and comparative analysis showed that BoWS was 84% identical to Arabidopsis thaliana putative water stress-induced protein (GenBank accession number AAM67282). Southern blot analysis indicated that BoWS was a low-copy gene. Northern blot analysis revealed that the expression of BoWS was upregulated by abscisic acid (ABA), mannitol, NaCl, drought, salicylic acid (SA) and hydrogen peroxide (H(2)O(2)). Our results indicate that BoWS is extremely related to the water-deficit stress in B. oleracea.     

Sodium (±)‐5‐bromo‐2‐(α‐hydroxypentyl) benzoate ameliorates pressure overload‐induced cardiac hypertrophy and dysfunction through inhibiting autophagy

Sodium (±)‐5‐bromo‐2‐(a‐hydroxypentyl) benzoate (generic name: brozopine, BZP) has been reported to protect against stroke‐induced brain injury and was approved for Phase II clinical trials for treatment of stroke‐related brain damage by the China Food and Drug Administration (CFDA). However, the role of BZP in cardiac diseases, especially in pressure overload‐induced cardiac hypertrophy and heart failure, remains to be investigated. In the present study, angiotensin II stimulation and transverse aortic constriction were employed to induce cardiomyocyte hypertrophy in vitro and in vivo, respectively, prior to the assessment of myocardial cell autophagy. We observed that BZP administration ameliorated cardiomyocyte hypertrophy and excessive autophagic activity. Further results indicated that AMP‐activated protein kinase (AMPK)‐mediated activation of the mammalian target of rapamycin (mTOR) pathway likely played a role in regulation of autophagy by BZP after Ang II stimulation. The activation of AMPK with metformin reversed the BZP‐induced suppression of autophagy. Finally, for the first time, we demonstrated that BZP could protect the heart from pressure overload‐induced hypertrophy and dysfunction, and this effect is associated with its inhibition of maladaptive cardiomyocyte autophagy through the AMPK‐mTOR signalling pathway. These findings indicated that BZP may serve as a promising compound for treatment of pressure overload‐induced cardiac remodelling and heart failure.     

A novel carbonyl reductase from Pichia stipitis for the production of ethyl (S)-4-chloro-3-hydroxybutanoate

An NADPH-dependent carbonyl reductase (PsCR) gene from Pichia stipitis was cloned. It contains an open reading frame of 849 bp encoding 283 amino acids whose sequence had less than 60% identity to known reductases that produce ethyl (S)-4-chloro-3-hydroxybutanoates (S-CHBE). When expressed in Escherichia coli, the recombinant PsCR exhibited an activity of 27 U/mg using ethyl 4-chloro-3-oxobutanoate (COBE) as a substrate. Reduction of COBE to (S)-CHBE by transformants in an aqueous mono-phase system for 18 h, gave a molar yield of 94% and an optical purity of the (S)-isomer of more than 99% enantiomeric excess.