Purification and Biochemical Characterization of Taxadiene Synthase from Bacillus koreensis and Stenotrophomonas maltophilia

Taxadiene synthase (TDS) is the rate-limiting enzyme of Taxol biosynthesis that cyclizes the geranylgeranyl pyrophosphate into taxadiene. Attenuating Taxol productivity by fungi is the main challenge impeding its industrial application; it is possible that silencing the expression of TDS is the most noticeable genomic feature associated with Taxol-biosynthetic abolishing in fungi. As such, the characterization of TDS with unique biochemical properties and autonomous expression that is independent of transcriptional factors from the host is the main challenge. Thus, the objective of this study was to kinetically characterize TDS from endophytic bacteria isolated from different plants harboring Taxol-producing endophytic fungi. Among the recovered 23 isolates, Bacillus koreensis and Stenotrophomonas maltophilia achieved the highest TDS activity. Upon using the Plackett–Burman design, the TDS productivity achieved by B. koreensis (18.1 µmol/mg/min) and S. maltophilia (14.6 µmol/mg/min) increased by ~2.2-fold over the control. The enzyme was purified by gel-filtration and ion-exchange chromatography with ~15 overall folds and with molecular subunit structure 65 and 80 kDa from B. koreensis and S. maltophilia, respectively. The chemical identity of taxadiene was authenticated from the GC-MS analyses, which provided the same mass fragmentation pattern of authentic taxadiene. The tds gene was screened by PCR with nested primers of the conservative active site domains, and the amplicons were sequenced, displaying a higher similarity with tds from T. baccata and T. brevifolia. The highest TDS activity by both bacterial isolates was recorded at 37–40 °C. The Apo-TDSs retained ~50% of its initial holoenzyme activities, ensuring their metalloproteinic identity. The activity of purified TDS was completely restored upon the addition of Mg2+, confirming the identity of Mg2+ as a cofactor. The TDS activity was dramatically reduced upon the addition of DTNB and MBTH, ensuring the implementation of cysteine-reactive thiols and ammonia groups on their active site domains. This is the first report exploring the autonomous robust expression TDS from B. koreensis and S. maltophilia with a higher affinity to cyclize GGPP into taxadiene, which could be a novel platform for taxadiene production as intermediary metabolites of Taxol biosynthesis.

From Structure to Atoms: Compression/Tension Systems to a Molecular Tensegrity

We reconsider macroscopic structure, including tensegrity structures, as ensembles of compression (C; repulsion) and tension (T; attraction) forces, and fit them to a triangular spectrum. Then, derivative structural analogy is made to the three classes of molecular bonding, as a bridge to microscopic structure. Basic molecular interactions and their “C/T” analogues are ionic bonds (with continuous compression/discontinuous tension), or metallic bonds (with both continuous tension and compression), or covalent bonds (with discontinuous compression/continuous tension—a tensegrity structure). The construction of tensegrity sculptures of particle interactions and the covalent molecules dihydrogen, methane, diborane, and benzene using tension and compression elements follows. We derived and utilized two properties in this analysis: 1) a “simplest tensegrity” subunit structure and 2) interpenetrating, discontinuous compressive members—tension members may also be discontinuous. This approach provides new artistic models for molecules and materials, and may inform future artistic, architectural, engineering and scientific endeavors.

The V-ATPase a3 Subunit: Structure, Function and Therapeutic Potential of an Essential Biomolecule in Osteoclastic Bone Resorption

This review focuses on one of the 16 proteins composing the V-ATPase complex responsible for resorbing bone: the a3 subunit. The rationale for focusing on this biomolecule is that mutations in this one protein account for over 50% of osteopetrosis cases, highlighting its critical role in bone physiology. Despite its essential role in bone remodeling and its involvement in bone diseases, little is known about the way in which this subunit is targeted and regulated within osteoclasts. To this end, this review is broadened to include the three other mammalian paralogues (a1, a2 and a4) and the two yeast orthologs (Vph1p and Stv1p). By examining the literature on all of the paralogues/orthologs of the V-ATPase a subunit, we hope to provide insight into the molecular mechanisms and future research directions specific to a3. This review starts with an overview on bone, highlighting the role of V-ATPases in osteoclastic bone resorption. We then cover V-ATPases in other location/functions, highlighting the roles which the four mammalian a subunit paralogues might play in differential targeting and/or regulation. We review the ways in which the energy of ATP hydrolysis is converted into proton translocation, and go in depth into the diverse role of the a subunit, not only in proton translocation but also in lipid binding, cell signaling and human diseases. Finally, the therapeutic implication of targeting a3 specifically for bone diseases and cancer is discussed, with concluding remarks on future directions.

Bioprocess Optimization of Glutathione Production by Saccharomyces Boulardii: Biochemical Characterization of Glutathione Peroxidase

The well-known probiotic GRAS Saccharomyces boulardii (CNCM I-745) was used for the first time to produce glutathione (GSH). The culture conditions affecting GSH biosynthesis were screened using a Plackett-Burman design (PBD). Analyzing the regression coefficients for 12 tested variables; 6 of them, including yeast extract, glucose, peptone and cysteine; temperature and agitation rate had a positive significant effect on GSH production with a maximum production of 192 mg/L. The impact of addition time of cysteine was investigated in 19 experiments during the growth time course (0-36 h), the best addition time was 8h post-inoculation producing 235 mg/L of GSH. The most significant variables were further explored at 5-levels using Central Composite Rotatable Design (CCRD), giving a maximum production of GSH (552 mg/L). Using baffled flasks, the GSH was increased to 730 mg/L, i.e 1.32-folds increment than obtained using CCRD. The two rate-limiting genes of GSH biosynthesis “γ-glutamyl cysteine synthetase (gsh1) and GSH-synthetase (gsh2) were amplified and sequenced to validate the GSH biosynthetic potency of S. boulardii. The sequences of genes showed 99% similarity with gsh1 and gsh2 genes of S. cerevisiae. Glutathione peroxidase was purified and characterized from S. boulardii with molecular mass and subunit structure of 80 kDa and 35 kDa as revealed from native and SDS-PAGE, ensuring its homodimeric identity. The activity of GPx was reduced by 2.5-folds upon demetallization confirming its metalloproteinic identity. The enzyme was strongly inhibited by hydroxylamine and DTNB, ensuring the implication of surface lysine and cysteine residues on the enzyme active site domains.

Reconstructed evolutionary history of the yeast septins Cdc11 and Shs1

Septins are GTP-binding proteins conserved across metazoans. They can polymerize into extended filaments and, hence, are considered a component of the cytoskeleton. The number of individual septins varies across the tree of life—yeast (Saccharomyces cerevisiae) has seven distinct subunits, a nematode (Caenorhabditis elegans) has two, and humans have 13. However, the overall geometric unit (an apolar hetero-octameric protomer and filaments assembled there from) has been conserved. To understand septin evolutionary variation, we focused on a related pair of yeast subunits (Cdc11 and Shs1) that appear to have arisen from gene duplication within the fungal clade. Either Cdc11 or Shs1 occupies the terminal position within a hetero-octamer, yet Cdc11 is essential for septin function and cell viability, whereas Shs1 is not. To discern the molecular basis of this divergence, we utilized ancestral gene reconstruction to predict, synthesize, and experimentally examine the most recent common ancestor (“Anc.11-S”) of Cdc11 and Shs1. Anc.11-S was able to occupy the terminal position within an octamer, just like the modern subunits. Although Anc.11-S supplied many of the known functions of Cdc11, it was unable to replace the distinct function(s) of Shs1. To further evaluate the history of Shs1, additional intermediates along a proposed trajectory from Anc.11-S to yeast Shs1 were generated and tested. We demonstrate that multiple events contributed to the current properties of Shs1: (1) loss of Shs1–Shs1 self-association early after duplication, (2) co-evolution of heterotypic Cdc11–Shs1 interaction between neighboring hetero-octamers, and (3) eventual repurposing and acquisition of novel function(s) for its C-terminal extension domain. Thus, a pair of duplicated proteins, despite constraints imposed by assembly into a highly conserved multi-subunit structure, could evolve new functionality via a complex evolutionary pathway.

The Central Benzodiazepine Receptor

Benzodiazepines have been used clinically now for more than a half century for the management of anxiety and other conditions. Despite their widespread use, only now are their mechanisms of action and their pharmacologic implications, especially with long-term use, beginning to be appreciated. In the central nervous system, benzodiazepines act on an allosteric site of the GABAA receptor to enhance the activity of this inhibitory neurotransmitter. The GABA receptor consists of 5 subunits, which can vary among 19 different subtypes, resulting in a large number of possible configurations for the GABA receptor. Subunit structure can affect the binding of benzodiazepines to the receptor and can alter the nature of the interaction between the benzodiazepine binding site and the GABA binding site, resulting in different levels of receptor activation and allosteric enhancement. Moreover, the distribution of these different subtypes within the central nervous system, with their varying levels of benzodiazepine efficacy, can mean that benzodiazepines can have differential effects among the different sites of the brain. Consequently, in may be possible to design novel drugs that favor particular subunit configurations and thus produce different pharmacologic profiles. Drugs acting at the benzodiazepine site can be agonists, enhancing the activity of GABA; antagonists, blocking the effect of benzodiazepines at the binding site; or inverse agonists, producing an effect antipodal to that of benzodiazepine agonists.

The Characteristics of New SSB Proteins from Metagenomic Libraries and Their Use in Biotech Applications

Single-stranded DNA binding proteins (SSBs) bind to single-stranded DNA in a sequence-independent manner to prevent formation of secondary structures and protect DNA from nuclease degradation. These ubiquitous proteins are present in prokaryotes, eukaryotes, and viruses, and play a pivotal role in the following major cellular processes: replication, recombination, and repair of genetic material. In DNA replication, SSB proteins specifically stimulate DNA polymerase, increase fidelity of DNA synthesis, assist the advance of DNA polymerase, and organize and stabilize replication forks. Here, we present our characterization of four SSB proteins of different origins. One of them was isolated from Clostridium sp. phage phiCP130 (SSB C1: 124 aa, Mr = 13,905). Three others (SSB M2: 136 aa, Mr = 15,009; SSB M3: 144 aa, Mr = 16,106; and SSB M5: 138 aa, Mr = 15,851) were isolated from metagenomics libraries. They show high similarity to SSB proteins from Caldanaerovirga acetigigens, Caldanaerobius fijiensis, and Fervidobacterium gondwanense. The recombinant proteins were overproduced in E. coli Rosetta (pRARE), except for SSB M5, which was overproduced in E. coli BL21. Proteins were purified using a metal-affinity chromatography as His-tagged fusion proteins. Electrophoretic mobility shift assay was used to examine their DNA binding activity with fluorescein-labeled oligonucleotide (dT40) used as a substrate. Thermal stability analysis revealed that they are stable at elevated temperatures, with the exception of SSB protein C1, which loses its activity above 65 °C. The other proteins are active at high temperatures, SSB M3 up to 85 °C, while SSB M2 and SSB M5 are active up to 98.7 °C. The subunit structure of proteins was analyzed by gel filtration on Superdex 75 column (AKTA). This allowed us to conclude that in solution, the analyzed proteins exist in oligomeric form, a feature which is characteristic of other SSB proteins. Purified SSB proteins were tested to improve specificity of PCR-based DNA amplification.