As a class of multifunctional biocatalysts, halohydrin dehalogenases are of great interest for the synthesis of chiral β-substituted alcohols and epoxides. There are less than 40 halohydrin dehalogenases with relatively clear catalytic functions, and most of them do not meet the requirements of scientific research and practical applications. Therefore, it is of great significance to excavate and identify more halohydrin dehalogenases. In the present study, a putative halohydrin dehalogenase (HHDH-Ra) from Rhodospirillaceae bacterium was expressed and its enzymatic properties were investigated. The HHDH-Ra gene was cloned into the expression host Escherichia coli BL21(DE3) and the target protein was shown to be soluble. Substrate specificity studies showed that HHDH-Ra possesses excellent specificity for 1,3-dichloro-2-propanol (1,3-DCP) and ethyl-4-chloro-3-hydroxybutyrate (CHBE). The optimum pH and temperature for HHDH-Ra with 1,3-DCP as the reaction substrate were 8.0 and 30 °C, respectively. HHDH-Ra was stable at pH 6.0-8.0 and maintained about 70% of its original activity after 100 h of treatment. The thermal stability results revealed that HHDH-Ra has a half-life of 60 h at 30 °C and 40 °C. When the temperature is increased to 50 °C, the enzyme still has a half-life of 20 h, which is much higher than that of the reported enzymes. To sum up, the novel halohydrin dehalogenase from Rhodospirillaceae bacterium possesses good temperature and pH stability as well as catalytic activity, and shows the potential to be used in the synthesis of chemical and pharmaceutical intermediates.
Escherichia coli/metabolism* ; Hydrolases/metabolism* ; Rhodospirillaceae ; Substrate Specificity

Halohydrin dehalogenase is of great significance for biodegradation of the chlorinated pollutants, and also serves as an important biocatalyst in the synthesis of chiral pharmaceutical intermediates. A putative halohydrin dehalogenase (HheTM) gene from Tistrella mobilis KA081020-065 was cloned and over-expressed in Escherichia coli BL21 (DE3). The recombinant enzyme was purified by Ni-NTA column and characterized. Gel filtration and SDS-PAGE analysis showed that the native form of HheTM was a tetramer. It exhibited the highest activity at 50 degrees C. The nature and pH of the buffer had a great effect on its activity. The enzyme maintained high stability under the alkaline conditions and below 30 degrees C. HheTM catalyzed the transformation of ethyl(S)-4-chloro-3-hydroxybutyrate in the presence of cyanide, to give ethyl (R)-4-cyano-3-hydroxybutyrate, a key intermediate for the synthesis of atorvastatin.
3-Hydroxybutyric Acid ; chemistry ; Bacterial Proteins ; genetics ; metabolism ; Cloning, Molecular ; Escherichia coli ; Hydrolases ; genetics ; metabolism ; Hydroxybutyrates ; chemistry ; Recombinant Proteins ; genetics ; metabolism ; Rhodospirillaceae ; enzymology ; genetics

OBJECTIVETo study the effects of extremely low frequency sinusoidal magnetic fields on hydrolysis of F(0)F(1)-ATPase and its mechanism.

METHODSThe F(0)F(1)-ATPases which was localized on the outer surface of chromatophores were prepared from the cells of Rhodospirillum rubrum and were exposed to 0.1 approximately 0.5 mT, 4.7 approximately 96.0 Hz magnetic fields.

RESULTSThe hydrolysis activity of F(0)F(1)-ATPase was stimulated by 0.5 mT, 4.7, 12.0, 60.0, 72.0, 84.0 and 96.0 Hz magnetic fields respectively and inhibited by 0.5 mT, 24.0 Hz magnetic field (P < 0.05); 0.3 mT, 4.7, 24.0 and 60.0 Hz magnetic fields also distinctly affected F(0)F(1)-ATPases activity respectively (P < 0.05), whereas 0.1 mT exposure caused no significant changes on that activity. When the hydrolysis activity of the F(0)F(1)-ATPases was inactivated by its inhibitor DCCD, the 0.5 mT, 24.0 Hz magnetic field still inhibited the hydrolysis activity of the F(0)F(1)-ATPase and 0.5 mT, 60.0 Hz magnetic field also had stimulating effects (P < 0.05).

CONCLUSIONThe effects of magnetic fields on the hydrolysis activity of the F(0)F(1)-ATPases depend on not only magnetic frequency but also magnetic intensity. The threshold of magnetic intensity is between 0.1 mT and 0.3 mT. F(0)F(1)-ATPases, especially F1-portion may be an end-point of magnetic fields.

Hydrolysis ; radiation effects ; Magnetic Fields ; adverse effects ; Proton-Translocating ATPases ; metabolism ; Rhodospirillum rubrum ; enzymology

Members of the ferric uptake regulator (Fur) protein family are bacterial transcriptional repressors that control iron uptake and storage in response to iron availability, thereby playing a crucial role in the maintenance of iron homeostasis. The fur null mutants of Pseudomonas aeruginosa could not be obtained because fur is an essential gene. In this study, We constructed a Fur inducibly expression strain Δfur/attB::PBAD-fur in order to study the effect of fur on the growth, biofilm formation, motilities and oxidative stress response of P. aeruginosa. The results showed that a low level of fur expression retarded the growth of P. aeruginosa at an iron-depleted condition, or under high concentration of iron, or in the presence of H2O2. Fur affected the biofilm formation and the motilities (swimming, twitching, and swarming) of strain PAO1. The production of pyoverdine is regulated by Fur. Interestingly, proteins from Magnetospirillum gryphiswaldense MSR-1, which shares homology with Fur, can partially recover the pyoverdine production of strain Δfur/attB::PBAD-fur. This study provides new clues for the prevention and treatment of P. aeruginosa infections.
Bacterial Proteins/genetics* ; Hydrogen Peroxide ; Magnetospirillum ; Pseudomonas aeruginosa/genetics* ; Repressor Proteins/genetics*

BACKGROUND AND OBJECTIVES: Poor homing efficiency is one of the major limitations of current stem cell therapy. Magnetic bionanoparticles (MPs) obtained from Magnetospirillum sp. AMB-1 have a lipid bilayer membrane and ferromagnetic properties. We evaluated a novel priming strategy using MPs to enhance the homing of transplanted progenitor cells to target tissue. MATERIALS AND METHODS: Effects of MP on proliferation, viability, and migration of late human endothelial progenitor cells (EPCs) were examined in vitro. Additionally, effects of MP on gene and protein expression related to survival and adhesion were evaluated. Homing and angiogenic efficiency of MP transferred late EPCs was evaluated in nude mouse hindlimb ischemia model. RESULTS: Below threshold concentration, MP transfer did not influence proliferation or survival of late EPCs, but enhanced migration and trans-endothelial migration of late EPCs toward magnet. Below threshold concentration, MP transfer did not influence gene and protein expression related to survival. In the mouse hindlimb ischemia model, late EPCs treated with high dose MP (5 ug/mL) showed enhanced homing of injected late EPCs in the ischemic limb by magnet, compared to low dose MP (1 ug/mL) treated late EPCs. In addition, high dose MP transferred EPC showed significantly better improvement of perfusion in ischemic limb compared to untreated EPC. CONCLUSION: MP transfer with magnet application can be a promising novel strategy to enhance homing efficacy and outcomes of current stem cell therapy.
Animals ; Extremities ; Hindlimb ; Humans ; Ischemia ; Lipid Bilayers ; Magnetics ; Magnetospirillum ; Magnets ; Membranes ; Mice ; Mice, Nude ; Nanoparticles ; Perfusion ; Phosphorylcholine ; Stem Cells ; Transplants

Magnetotactic bacteria (MTB), a group of phylogenetically diverse organisms that use their unique intracellular magnetosome organelles to swim along the Earth's magnetic field, play important roles in the biogeochemical cycles of iron and sulfur. Previous studies have revealed that the bacterial actin protein MamK plays essential roles in the linear arrangement of magnetosomes in MTB cells belonging to the Proteobacteria phylum. However, the molecular mechanisms of multiple-magnetosome-chain arrangements in MTB remain largely unknown. Here, we report that the MamK filaments from the uncultivated 'Candidatus Magnetobacterium casensis' (Mcas) within the phylum Nitrospirae polymerized in the presence of ATP alone and were stable without obvious ATP hydrolysis-mediated disassembly. MamK in Mcas can convert NTP to NDP and NDP to NMP, showing the highest preference to ATP. Unlike its Magnetospirillum counterparts, which form a single magnetosome chain, or other bacterial actins such as MreB and ParM, the polymerized MamK from Mcas is independent of metal ions and nucleotides except for ATP, and is assembled into well-ordered filamentous bundles consisted of multiple filaments. Our results suggest a dynamically stable assembly of MamK from the uncultivated Nitrospirae MTB that synthesizes multiple magnetosome chains per cell. These findings further improve the current knowledge of biomineralization and organelle biogenesis in prokaryotic systems.
Actins ; chemistry ; metabolism ; Adenosine Triphosphate ; metabolism ; Bacteria ; classification ; metabolism ; Bacterial Proteins ; chemistry ; metabolism ; Magnetospirillum ; classification ; metabolism ; Nucleotides ; metabolism ; Phylogeny ; Substrate Specificity