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Abstract l-2-Keto-3-deoxyfuconate 4-dehydrogenase (l-KDFDH) catalyzes the NAD+-dependent oxidization of l-2-keto-3-deoxyfuconate (l-KDF) to l-2,4-diketo-3-deoxyfuconate (l-2,4-DKDF) in the non-phosphorylating l-fucose pathway from bacteria, and its substrate was previously considered to be the acyclic α-keto form of l-KDF. On the other hand, BDH2, a mammalian homolog with l-KDFDH, functions as a dehydrogenase for cis-4-hydroxy-l-proline (C4LHyp) with the cyclic structure. We found that l-KDFDH and BDH2 utilize C4LHyp and l-KDF, respectively. Therefore, to elucidate unique substrate specificity at the atomic level, we herein investigated for the first time the crystal structures of l-KDFDH from Herbaspirillum huttiense in the ligand-free, l-KDF and l-2,4-DKDF, d-KDP (d-2-keto-3-deoxypentonate; additional substrate), or l-2,4-DKDF and NADH bound forms. In complexed structures, l-KDF, l-2,4-DKDF, and d-KDP commonly bound as a α-furanosyl hemiketal. Furthermore, l-KDFDH showed no activity for l-KDF and d-KDP analogs without the C5 hydroxyl group, which form only the acyclic α-keto form. The C1 carboxyl and α-anomeric C2 hydroxyl groups and O5 oxygen atom of the substrate (and product) were specifically recognized by Arg148, Arg192, and Arg214. The side chain of Trp252 was important for hydrophobically recognizing the C6 methyl group of l-KDF. This is the first example showing the physiological role of the hemiketal of 2-keto-3-deoxysugar acid.

ABSTRACTIn tsunami inundations or slope disasters of heavy rain, a lot of floating debris or driftwood logs are included in the flows. The damage to structures from solid body impacts is more severe than the damage from the water pressure. In order to study free-surface flows that include floating debris, developing a high-accurate simulation code of free-surface flows with high performance for large-scale computations is desired. We propose the single-phase free-surface flow model based on the cumulant lattice Boltzmann method coupled with a particle-based rigid body simulation. The discrete element method calculates the contact interaction between solids. An octree-based AMR (Adaptive Mesh Refinement) method is introduced to improve computational accuracy and time-to-solution. High-resolution grids are assigned near the free surfaces and solid boundaries. We conducted two kinds of tsunami flow experiments in the 15 and 70 m water tanks at Hachinohe Institute of Technology and Kobe University to validate the accuracy of the proposed model. The simulation results have shown good agreement with the experiments for the drifting speed, the number of trapped wood pieces, and the stacked angles.

Abstract Xylitol dehydrogenase (XDH) catalyzes the NAD+-dependent oxidization of xylitol into d-xylulose, and belongs to a zinc-dependent medium-chain dehydrogenase/reductase family. This protein family consists of enzymes with one or two zinc atoms per subunit, among which catalytic zinc is necessary for the activity. Among many XDHs from yeast and fungi, XDH from Pichia stipitis is one of the key enzymes for bioethanol production by lignocellulosic biomass, and possesses only a catalytic zinc atom. Despite its importance in bioindustry, a structural data of XDH has not yet been available, and little insight into the role of a second zinc atom in this protein family is known. We herein report the crystal structure of XDH from P. stipitis using a thermostabilized mutant. In the refined structure, a second zinc atom clearly coordinated with four artificially introduced cysteine ligands. Homologous mutations in XDH from Saccharomyces cerevisiae also stabilized and enhanced activity. The substitution of each of the four cysteine ligands with an aspartate in XDH from Schizosaccharomyces pombe contributed to the significantly better maintenance of activity and thermostability than their substitution with a serine, providing a novel hypothesis for how this zinc atom was eliminated.

Abstract Adsorbed molecular films, referred to as boundary films in tribology, are widely used in various industrial products as a keyway for surface functionalisation, such as lubricity, wettability, and adhesion. Because boundary films are thin nanometre-scale molecular layers and can easily be removed, their formation process cannot be elucidated in detail. In this study, to analyse the growth dynamics of boundary films, the film thickness and molecular orientation of the boundary film of a fatty acid used as an additive in rolling contact as mechanical stimuli were measured in situ. The measurements were performed on simple test lubricants, which were composed of n-hexadecane and stearic acid, at rolling tribological condition between steel and glass (or sapphire) surfaces by ultrathin film interferometry combined with sum-frequency generation spectroscopy according to a unique protocol. The results quantitatively demonstrate shear-induced boundary film formation. The insight gained from these results is anticipated to enable the formulation of high-performance lubricant additives to further reduce friction loss and high-performance glues that can be freely designed for removability.

Introduction: There is a growing momentum for the collaboration between multiple disciplines for the prevention and treatment of skeletal-related events (SREs) in patients with metastatic spinal tumors. However, the effectiveness of multidisciplinary approaches remains unclear. Hence, we conducted an exploratory study to examine the impact of liaison treatment for metastatic spinal tumor (LMST) on the prevention of SREs among patients with a metastatic spinal tumor. Methods: This study was an exploratory interrupted time series conducted in a single medical center. Overall, 1,043 patients with a metastatic spinal tumor diagnosed between January 2011 and December 2020 were included. The LMST was implemented in January 2014. The LMST team consisted of the orthopedic surgery, thoracic surgery, breast and thyroid surgery, clinical oncology, urology, and radiology departments. Monthly joint conferences were held for patients with spinal instability, and the incidence of SRE was measured at 6-month intervals. Results: Throughout the study period, we identified 66 SRE incidences. After the implementation of the LMST, a level change of −5.2% (95% confidence interval [CI]: −11.7 to 1.3, p = 0.11) was observed. Subsequently, a post-implementation trend change of −0.3% (95% CI: −2.0 to 1.5, p = 0.75) beyond the baseline was noted. Conclusions: We suggest both immediate and gradual effects of the introduction of the LMST on deterring the development of SREs. Our results support the global trend of introducing a multidisciplinary approach for the treatment of metastatic spinal tumors.

Ionic liquids (ILs) are being considered as novel lubricant additives to improve the friction and anti-wear characteristics of sliding surfaces. Because ILs consist of only cations and anions, their physical and chemical properties can be easily tailored by modifying their combination. These features enable to design of specific ILs for a given tribological system. However, there are few reports on the effect of ILs used as lubricant additives on the friction and anti-wear characteristics of sliding surfaces. Moreover, it is necessary to investigate the synergism between ILs and other lubricant additives to achieve high practical efficiency. Therefore, in this study, two ILs with different cations but similar anions were used in combination with an anti-wear additive, zinc dialkyldithiophosphate (ZDDP), and the resultant wear and friction characteristics of the worn surfaces were analyzed. We found that the tribological performance under steel/steel sliding conditions was dependent on the chemical composition of the ILs. In addition, mixed ZDDP and IL solutions exhibited lower friction and wear when compared to ZDDP alone. It is considered that the excellent lubrication and frictional performance observed with the mixed lubricant solutions is due to the formation of tribofilms comprising of ZDDP and IL on the sliding surface.

Seiya Watanabe et al. report the crystal structures of two distinct members of the Aconitase X subfamily, which contain [Fe-S] clusters different from other aconitases. This study provides insight into the molecular evolution of the aconitase superfamily.

When various sliding components of combustion engines protected with hard coatings are slid with automotive lubricants, unexpected interfacial phenomena may occur because typical automotive lubricants have been formulated with additives suitable for steel/steel sliding components. Therefore, understanding the effects of hard coatings on the performance of lubricant additives is crucial for further improving the lubrication properties of fully formulated oils through the optimization of additive formulations for hard coatings. In this study, friction tests were performed with a steel surface against steel surfaces coated with two ceramic materials and two diamond-like carbon materials under lubrication with a fully formulated oil. Atomic force microscopy and X-ray photoelectron spectroscopic (XPS) investigation of the steel surfaces after the friction tests revealed that the tribofilms comprised a bottom layer composed of calcium compounds and a top layer composed of phosphorous, zinc, and sulfur compounds. The thickness of each layer, which is closely related to the wear property, depends on the hard coating material. Moreover, XPS analysis focusing on the molybdenum element suggested that rate of chemical kinetics to form MoS2 differs among the hard coating materials, and dominantly affects the frictional property.

Introduction: Due to the increase in osteoporosis accompanying the aging society in Japan, osteoporotic vertebral fractures (OVFs) are increasing. Percutaneous vertebral augmentation (PVA) has been widely used for OVFs because it reduces pain immediately with less invasiveness. Re-collapse of vertebral body after PVA is a rare, but important, complication. Once the re-collapse has occurred, patients should undergo an additional invasive salvage surgery. Methods: We treated 5 patients with re-collapse after PVA in our hospital. For re-collapse after PVA, we performed anterior column reconstruction with video-assisted thoracoscopic surgery (VATS), posterior fixation with percutaneous pedicle screws (PPSs) and minimally invasive spine stabilization (MISt). Results: The mean postoperative follow-up was at 62.8 months. At the final follow-up, the patients were free of low back pain, and bony union was achieved in all cases. The postoperative correction loss was 6 degrees. Perioperative complications included aspiration pneumonia in one patient and bone fracture of an adjacent vertebral body in two patients. There were no reoperation cases. Conclusions: We perform minimally invasive combined anterior and posterior surgery with VATS for re-collapse after PVA. This procedure is useful in elderly patients with less reserve capacity.

Introduction: The cancer board system employed at many hospitals and treatment centers involves multidisciplinary healthcare teams, including physicians, and the timing of treatment generally follows that of a consultation model. Thus, it is difficult to detect spinal metastases using the current implementation of the cancer board system, which can lead to delays in treatment. A new multidisciplinary treatment strategy for patients with metastatic spinal tumors was designed, and 745 patients were treated based on this strategy. Methods: In the first 5 years using the liaison treatment approach, 745 patients were diagnosed with metastatic spinal tumor. Tumors were discovered before a skeletal-related event (SRE) in 704 patients and after an SRE in 41 patients. We conducted our analysis in two patient groups: those with and without an SRE at the time of treatment initiation. Results: In most patients, the average spinal instability neoplastic score was 5.2, which indicates that we were able to detect the spinal tumor before a significant breakdown of the spinal support system. Ninety-five percent of patients were classified according to the Frankel grade classification during their initial diagnosis, and many patients initially underwent treatment before the onset of paralysis. Of patients with an SRE, 33% were Frankel grade E, indicating that approximately half were paralyzed at initial diagnosis. The median survival duration was prolonged by approximately 9 months in patients without an SRE compared with those with an SRE. Conclusions: Orthopedic spine surgeons are responsible for maintaining activities of daily living, improving quality of life, and prolonging life expectancy in patients with metastatic spinal tumors. The results of this study revealed that the liaison treatment system for metastatic spinal tumors has made it possible to successfully prevent SREs without neurological deficits and to prolong survival.

Background and Objectives: There have been numerous advances in spine surgery for metastatic spinal tumors, and minimally invasive spine stabilization (MISt) is becoming increasingly popular in Japan. MISt is a minimally invasive fixation procedure that temporarily stabilizes the spine, thereby reducing pain, preventing pathological fractures, and improving activities of daily living at an early stage. MISt may be useful given the recent shift toward outpatient cancer treatment. Materials and Methods: This study enrolled 51 patients with metastatic spinal tumors who underwent surgery using MISt between December 2013 and October 2020. The Spinal Instability Neoplastic Score, an assessment of spinal instability, was used to determine the indication for surgery, and the Epidural Spinal Cord Compression scale was used for additional decompression. Results: The patients comprised 34 men and 17 women, and the mean age at surgery was 68.9 years. The mean postoperative follow-up period was 20.8 months, and 35 of 51 patients (67%) had died by the last survey. The mean operative time was 159.8 min, mean blood loss was 115.7 mL, and mean time to ambulation was 3.2 days. No perioperative complications were observed, although two patients required refixation surgery. Preoperatively, 37 patients (72.5%) were classified as Frankel grade E. There were no cases of postoperative exacerbation, and six patients showed improvement of one or more Frankel grades after surgery. The median duration of patient survival was about 22.0 months. Patients with breast, prostate, renal, and thyroid cancers had a good prognosis, whereas those with gastrointestinal and head and neck cancers had a poor prognosis. Conclusions: MISt can benefit patients who are ineligible for conventional, highly invasive surgery and is also suitable because cancer treatment is increasingly performed on an outpatient basis. Furthermore, choosing the right surgery for the right patient at the right time can significantly affect life expectancy.

Agricultural waste biomass has already been transferred to bioethanol and used as energy related products, although many issues such as efficiency and productivity still exist to be overcome. In this study, the protein engineering was applied to generate enzymes with completely reversed coenzyme specificity and developed recombinant yeasts containing those engineered enzymes for construction of an efficient biomass-ethanol conversion system. Recombinant yeasts were constructed with the genes encoding a wild type xylose reductase (XR) and the protein engineered xylitol dehydrogenase (XDH) (with NADP) of Pichia stipitis. These recombinant yeasts were characterized based on the enzyme activity and fermentation ability of xylose to ethanol. The protein engineered enzymes were expressed significantly in Saccharomyces cerevisiae as judged by the enzyme activity in vitro. Ethanol fermentation was measured in batch culture under anaerobic conditions. The significant enhancement was found in Y-ARS strain, in which NADP+-dependent XDH was expressed; 85% decrease of unfavorable xylitol excretion with 26% increased ethanol production, when compared with the reference strain expressing the wild–type XDH.

The effects of the molecular structure of zinc dialkyldithiophosphates (ZDDPs) on the tribological properties of a hydrogenated amorphous carbon (a-C:H) film under boundary lubrication were investigated. Friction tests were performed at a-C:H/a-C:H and steel/steel contacts under lubrication with poly-alpha olefin (PAO) and PAO containing five types of ZDDPs with different alkyl groups (primary-C4 ZDDP, secondary-C4 ZDDP, primary-C6 ZDDP, primary-C8 ZDDP and primary-C12 ZDDP). All our results suggest that the structure of the alkyl chain of ZDDPs strongly influence the tribological properties of the a-C:H/a-C:H and steel/steel tribopairs lubricated with ZDDP solutions.

Ionic liquids have potential for use as novel high-performance lubricants because of their attractive characteristics such as low volatility, high-thermal stability, and oxidation stability. It is known that ionic liquids exhibit excellent lubricity for metals because of halogen constituents in their molecular structure. However, occurrence of corrosive damage on the contacting surfaces lubricated with the ionic liquids has also been reported. To prevent damage due to corrosion, it is necessary to use halogen-free ionic liquids whose lubricity may be inferior compared with that of halogen-containing ionic liquids. In this study, the lubricity of halogen-free ionic liquids 1-butyl-3-methylimidazolium dicyanamide ([BMIM][DCN]) and 1-butyl-3-methylimidazolium tricyanomethane ([BMIM][TCC]) was evaluated by using a reciprocating sliding friction and wear tester (SRV Optimol)) using an oscillating steel cylinder on H-free DLC disk test configuration under boundary lubrication conditions. The SRV test results showed that H-free DLC with [BMIM][TCC] at 50N exhibited superior lubricity than that with [BMIM][DCN] at the same load. In order to understand the observed, the worn surfaces of test specimens were analyzed by using Raman spectroscopy, the friction coefficient of the film by atomic force microscopy (AFM), and the chemical composition by time of flight secondary ion mass spectrometry (TOF-SIMS). From Raman spectroscopic analysis results, graphitization of the transfer films of both [BMIM][DCN] and [BMIM][TCC] did not occur. From AFM results, the topography of [BMIM][TCC] was smooth as compared with that of [BMIM][DCN]. Thus, it was considered that the surface roughness affected the lubricity. In addition, the tribofilm derived from [BMIM][TCC] also affected the lubricity because it showed low friction coefficient on the flat position. From ToF-SIMS results, it was considered that [BMIM][DCN] itself adsorbed on H-free DLC surface. On the other hand, [BMIM][TCC] is thermally decomposed by friction and generated isolated CN and covering the H-free DLC surface. The tribofilm formed by [BMIM][TCC] exhibited lower friction coefficient than that by [BMIM][DCN].

The tribological properties of diamond-like carbon (DLC) films that were lubricated with an environmentally friendly ester-based oil were investigated. Sliding tests were performed on two types of DLC films, hydrogenated amorphous carbon (a-C:H) and tetrahedral amorphous carbon (ta-C) films, using a steel cylinder that was in contact with a steel disk coated with either a-C:H or ta-C under boundary lubrication. To compare the tribological properties of ester-based oils and other based oils, Trimethylolpropane (TMP) ester, polypropylene glycol (PPG), and poly-alpha olefin (PAO) were used as the test lubricants. The ta-C film that was lubricated with TMP ester exhibited the ultralow friction coefficient of 0.015; however, the a-C:H film that was lubricated with TMP ester did not exhibit the ultralow friction. On the other hand, results obtained by a quartz-crystal microbalance with dissipation indicated that the adsorption properties of based oils depended on the type of sensor material (carbon or iron) and the type of based oil used in the measurements. Moreover, the frequency change of based oils increased with decreases in the average frictional coefficients of the steel-steel tribopair and the DLC-steel tribopair. Results of surface analysis indicated that TMP ester formed tribofilms on the ta-C surfaces, which exhibited high viscosity and low friction. All these results suggest that the combination of the ta-C film and TMP ester is the new ultra-low-friction and environmentally friendly sliding system.

The gene encoding the Saccharomyces cerevisiae phospholipid deacylation enzyme, phospholipase B (ScPLB1), was successfully expressed in E. coli. The enzyme (Scplb1p) was engineered to have a histidine-tag at the C-terminal end and was purified by metal (Ni) affinity chromatography. Enzymatic properties, optimal pH, and substrate specificity were similar to those reported previously. For example, deacylation activity was observed in acidic pH in the absence of Ca2+ and was additive in neutral pH in the presence of Ca2+, and the enzyme had the same substrate priority as reported previously, with the exception of PE, suggesting that yeast phospholipase B could be produced in its native structure in bacterial cells. Scplb1p retained transacylation activity in aqueous medium, and esterified lysophosphatidylcholine with free fatty acid to form phosphatidylcholine in a non-aqueous, glycerin medium. We propose that phospholipase B could serve as an additional tool for in vitro enzyme-mediated phospholipid synthesis.

Agricultural waste biomasshas already been transferred to bioethanol and used as energy related products, although many issues such as efficiency and productivity still exist to be overcome. In this study, the protein engineering was applied to generate enzymes with completely reversed coenzyme specificity and developed recombinant yeasts containing those engineered enzymes for construction of an efficient biomass-ethanol conversion system. Recombinant yeasts were constructed with the genes encoding a wild type xylose reductase (XR)and the protein engineered xylitol dehydrogenase (XDH)(with NADP) of Pichiastipitis. These recombinant yeasts were characterized based on the enzyme activity and fermentation ability of xylose to ethanol. The protein engineered enzymes were expressed significantly in Saccharomycescerevisiaeas judged by the enzyme activity in vitro. Ethanol fermentation was measured in batch culture under anaerobic conditions. The significant enhancement was found in Y-ARSstrain, in which NADP+-dependentXDH was expressed; 85% decrease of unfavorable xylitol excretion with 26% increased ethanol production, when compared with the reference strain expressing the wild–type XDH.

l-Ornithine cyclodeaminase (OCD) is involved in l-proline biosynthesis and catalyzes the unique deaminating cyclization of l-ornithine to l-proline via a Δ1-pyrroline-2-carboxyrate (Pyr2C) intermediate. Although this pathway functions in only a few bacteria, many archaea possess OCD-like genes (proteins), among which only AF1665 protein (gene) from Archaeoglobus fulgidus has been characterized as an NAD+-dependent l-alanine dehydrogenase (AfAlaDH). However, the physiological role of OCD-like proteins from archaea has been unclear. Recently, we revealed that Pyr2C reductase, involved in trans-3-hydroxy-l-proline (T3LHyp) metabolism of bacteria, belongs to the OCD protein superfamily and catalyzes only the reduction of Pyr2C to l-proline (no OCD activity) [FEBS Open Bio (2014) 4, 240–250]. In this study, based on bioinformatics analysis, we assumed that the OCD-like gene from Thermococcus litoralis DSM 5473 is related to T3LHyp and/or proline metabolism (TlLhpI). Interestingly, TlLhpI showed three different enzymatic activities: AlaDH; N-methyl-l-alanine dehydrogenase; Pyr2C reductase. Kinetic analysis suggested strongly that Pyr2C is the preferred substrate. In spite of their similar activity, TlLhpI had a poor phylogenetic relationship to the bacterial and mammalian reductases for Pyr2C and formed a close but distinct subfamily to AfAlaDH, indicating convergent evolution. Introduction of several specific amino acid residues for OCD and/or AfAlaDH by site-directed mutagenesis had marked effects on both AlaDH and Pyr2C reductase activities. The OCC_00387 gene, clustered with the TlLhpI gene on the genome, encoded T3LHyp dehydratase, homologous to the bacterial and mammalian enzymes. To our knowledge, this is the first report of T3LHyp metabolism from archaea.

trans-4-Hydroxy-l-proline (T4LHyp) and trans-3-hydroxy-l-proline (T3LHyp) occur mainly in collagen. A few bacteria can convert T4LHyp to α-ketoglutarate, and we previously revealed a hypothetical pathway consisting of four enzymes at the molecular level (J Biol Chem (2007) 282, 6685–6695; J Biol Chem (2012) 287, 32674–32688). Here, we first found that Azospirillum brasilense has the ability to grow not only on T4LHyp but also T3LHyp as a sole carbon source. In A. brasilense cells, T3LHyp dehydratase and NAD(P)H-dependent Δ1-pyrroline-2-carboxylate (Pyr2C) reductase activities were induced by T3LHyp (and d-proline and d-lysine) but not T4LHyp, and no effect of T3LHyp was observed on the expression of T4LHyp metabolizing enzymes: a hypothetical pathway of T3LHyp → Pyr2C → l-proline was proposed. Bacterial T3LHyp dehydratase, encoded to LhpH gene, was homologous with the mammalian enzyme. On the other hand, Pyr2C reductase encoded to LhpI gene was a novel member of ornithine cyclodeaminase/μ-crystallin superfamily, differing from known bacterial protein. Furthermore, the LhpI enzymes of A. brasilense and another bacterium showed several different properties, including substrate and coenzyme specificities. T3LHyp was converted to proline by the purified LhpH and LhpI proteins. Furthermore, disruption of LhpI gene from A. brasilense led to loss of growth on T3LHyp, d-proline and d-lysine, indicating that this gene has dual metabolic functions as a reductase for Pyr2C and Δ1-piperidine-2-carboxylate in these pathways, and that the T3LHyp pathway is not linked to T4LHyp and l-proline metabolism.

l-hydroxyproline (l-Hyp: trans-4-hydroxy-l-proline) is commonly found at high concentrations in connective tissue proteins such as collagen. It is a remarkably useful molecular marker because variation in the level of l-Hyp is associated with various diseases. Recently, the novel enzymes l-hydroxyproline epimerase and d-hydroxyproline dehydrogenase were isolated from bacteria. In this study, a novel electrochemical biosensor for l-Hyp was constructed using these two enzymes. l-hydroxyproline epimerase epimerized l-Hyp to d-hydroxyproline (d-Hyp: cis-4-hydroxy-d-proline), and d-Hyp was oxidized with the reaction catalyzed by d-hydroxyproline dehydrogenase and mediated by ferrocene. We found that the sensor could determine l-Hyp concentrations of 10–100 μM with high-selectivity.

The friction coefficients of ionic liquids were evaluated by many investigations. Most investigations used fluorine-based ionic liquids as lubricants. However, these ionic liquids produce the corrosion wear. This investigation focuses on the use of cyano-based ionic liquids as lubricants. Compared to fluorine-based ionic liquids, cyano-based ionic liquids exhibit high friction coefficients against steel material. This work examines how the friction coefficients of cyano-based ionic liquids are influenced by the type of sliding material used (AISI 52100, TiO2, and tetrahedral amorphous carbon). TiO2 lubricated with 1-ethyl-3-methylimidazolium tricyanomethanide, and ta-C lubricated with 1-butyl-1methylpyrrolidinium tetracyanoborate exhibited very low friction coefficients, smaller than fluorine-based ionic liquids. Time-of-Flight Secondary Ion Mass Spectrometry analysis showed that anions adsorb onto the worn surface, suggesting that anion adsorption is a critical parameter influencing friction coefficients. Quadrupole Mass Spectrometry measurements revealed that cations decompose on the nascent surface, preventing adsorption on the worn surface. These results suggest that low friction coefficients require the decomposition of cations and adsorption of anions. The reactivity of nascent surface changes with the sliding material used due to varying catalytic activity of the nascent surfaces.

Opines, in particular nopaline and octopine, are specific compounds found in crown gall tumor tissues induced by infections with Agrobacterium species, and are synthesized by well-studied NAD(P)H-dependent dehydrogenases (synthases), which catalyze the reductive condensation of α-ketoglutarate or pyruvate with L-arginine. The corresponding genes are transferred into plant cells via a tumor-inducing (Ti) plasmid. In addition to the reverse oxidative reaction(s), the genes noxB-noxA and ooxB-ooxA are considered to be involved in opine catabolism as (membrane-associated) oxidases; however, their properties have not yet been elucidated in detail due to the difficulties associated with purification (and preservation). We herein successfully expressed Nox/Oox-like genes from Pseudomonas putida in P. putida cells. The purified protein consisted of different α-, β-, and γ-subunits encoded by the OdhA, OdhB, and OdhC genes, which were arranged in tandem on the chromosome (OdhB-C-A), and exhibited dehydrogenase (but not oxidase) activity toward nopaline in the presence of artificial electron acceptors such as 2,6-dichloroindophenol. The enzyme contained FAD, FMN, and [2Fe-2S]-iron sulfur as prosthetic groups. On the other hand, the gene cluster from Bradyrhizobium japonicum consisted of OdhB1-C-A-B2, from which two proteins, OdhAB1C and OdhAB2C, appeared through the assembly of each β-subunit together with common α- and γ-subunits. A poor phylogenetic relationship was detected between OdhB1 and OdhB2 in spite of them both functioning as octopine dehydrogenases, which provided clear evidence for the acquisition of novel functions by "subunit-exchange". To the best of our knowledge, this is the first study to have examined flavin-containing opine dehydrogenase.

Proline racemase (ProR) is a member of the pyridoxal 5'-phosphate-independent racemase family, and is involved in the Stickland reaction (fermentation) in certain clostridia as well as the mechanisms underlying the escape of parasites from host immunity in eukaryotic Trypanosoma. Hydroxyproline epimerase (HypE), which is in the same protein family as ProR, catalyzes the first step of the trans-4-hydroxy-L-proline metabolism of bacteria. Their substrate specificities were previously considered to be very strict, in spite of similarities in their structures and catalytic mechanisms, and no racemase/epimerase from the ProR superfamily has been found in archaea. We here characterized the ProR-like protein (OCC_00372) from the hyperthermophilic archaeon, Thermococcus litoralis (TlProR). This protein could reversibly catalyze not only the racemization of proline, but also the epimerization of 4-hydroxyproline and 3-hydroxyproline with similar kinetic constants. Among the four (putative) ligand binding sites, one amino acid substitution was detected between TlProR (tryptophan at the position of 241) and natural ProR (phenylalanine). The W241F mutant showed a significant preference for proline over hydroxyproline, suggesting that this (hydrophobic and bulky) tryptophan residue played an importance role in the recognition of hydroxyproline (more hydrophilic and bulky than proline), and substrate specificity for hydroxyproline was evolutionarily acquired separately between natural HypE and ProR. A phylogenetic analysis indicated that such unique broad substrate specificity was derived from an ancestral enzyme of this superfamily.

Background: Here, we assessed a new trajectory and insertion torque for the placement of a long lateral mass screw (LLMS) that offers stronger posterior fixation versus a shorter lateral mass screw (LMS) in the posterior cervical spine. We report a short technical note of the insertion torque of LLMS. Methods: The insertion trajectory/torque was evaluated in 30 patients (10 males and 20 females) undergoing posterior cervical LLMS fusions (2021-2023). Patients averaged 65 years of age. Pathology included eight cervical spine injuries, ten cord injuries, four dislocations/fractures, and eight other entities. Variables studied included the length of the LLMS inserted from C3-7, screw deviation rates, insertion torque, and adverse events. Results: A total of 146 screws were inserted: 11 pedicle screws (PSs) and 135 LLMS. The average insertion torque was 105.9 cNm for PS and 64.9 cNm for LLMS. As the screw lengthened by 1 mm, the insertion torque increased by approximately 4.4 cNm. Conclusion: Here, we documented that the insertion torque of LLMS was 66.1 cNm, greater than the 51.0 cNm for LMS, which should provide stronger posterior cervical fixation. [ABSTRACT FROM AUTHOR]