The Mycobacterium tuberculosis Cell Wall: An Alluring Drug Target for Developing Newer Anti-TB Drugs-A Perspective.

The Mycobacterium cell wall is a capsule-like structure comprising of various layers of biomolecules such as mycolic acid, peptidoglycans, and arabinogalactans, which provide the Mycobacteria a sort of cellular shield. Drugs like isoniazid, ethambutol, cycloserine, delamanid, and pretomanid inhibit cell wall synthesis by inhibiting one or the other enzymes involved in cell wall synthesis. Many enzymes present across these layers serve as potential targets for the design and development of newer anti-TB drugs. Some of these targets are currently being exploited as the most druggable targets like DprE1, InhA, and MmpL3. Many of the anti-TB agents present in clinical trials inhibit cell wall synthesis. The present article covers a systematic perspective of developing cell wall inhibitors targeting various enzymes involved in cell wall biosynthesis as potential drug candidates for treating Mtb infection.

CTBP1 links metabolic syndrome to polycystic ovary syndrome through interruption of aromatase and SREBP1.

Some patients with polycystic ovarian syndrome (PCOS) suffered from metabolic syndrome (MetS) including dyslipidemia, hyperinsulinism, but the underlying mechanism is unclear. Although C-terminal Binding Protein 1 (CTBP1) is a transcriptional co-repressor frequently involved in hormone secretion disorders and MetS-associated diseases, the role of CTBP1 in PCOS is rarely reported. In the present study, we found that CTBP1 expression was significantly elevated in primary granulosa cells (pGCs) derived from the PCOS with MetS patients and was positively associated with serum triglyceride, but negatively correlated with serum estradiol (E2) or high-density lipoprotein. Mechanistic study suggested that CTBP1 physically bound to the promoter II of cytochrome P450 family 19 subfamily A member 1 (CYP19A1) to inhibit the aromatase gene transcription and expression, resulting in the reduced E2 synthesis. Moreover, CTBP1 interacted with the phosphorylated SREBP1a at S396 in nuclei, leading to the FBXW7-dependent protein degradation, resulting in the reduced lipid droplets formation in pGCs. Therefore, we conclude that CTBP1 in GCs dysregulates the synthesis of steroid hormones and lipids through suppression of aromatase expression and promotion of SREBP1a protein degradation in PCOS patients, which may offer some fresh insights into the potential pathological mechanism for this tough disease.

Validation of shikimate dehydrogenase as the herbicidal target of drupacine and screening of target-based compounds with high herbicidal activity.

The discovery of new targets and lead compounds is the key to developing new pesticides. The herbicidal target of drupacine has been identified as shikimate dehydrogenase (SkDH). However, the mechanism of interaction between them remains unclear. This study found that drupacine specifically binds to SkDH with a dissociation equilibrium constant (KD) of 8.88 µM and a Kd value of 2.15 µM, as confirmed by surface plasmon resonance and microscale thermophoresis. Site-directed mutagenesis coupled with fluorescence quenching analysis indicated that residue THR431 was the key amino acid site for drupacine binding to SkDH. Nine compounds with the best binding ability to SkDH were identified by virtual screening from about 120,000 compounds. Among them, compound 8 showed the highest inhibition rate with values of 41.95% against SkDH, also exhibiting the strongest herbicidal activity. This research identifies a novel potential target SkDH and a candidate lead compound with high herbicidal activity for developing new herbicides.

Genome-wide characterization and expression analysis of the CINNAMYL ALCOHOL DEHYDROGENASE gene family in Triticum aestivum.

BACKGROUND: CINNAMYL ALCOHOL DEHYDROGENASE (CAD) catalyzes the NADPH-dependent reduction of cinnamaldehydes into cinnamyl alcohols and is a key enzyme found at the final step of the monolignol pathway. Cinnamyl alcohols and their conjugates are subsequently polymerized in the secondary cell wall to form lignin. CAD genes are typically encoded by multi-gene families and thus traditionally organized into general classifications of functional relevance. RESULTS: In silico analysis of the hexaploid Triticum aestivum genome revealed 47 high confidence TaCAD copies, of which three were determined to be the most significant isoforms (class I) considered bone fide CADs. Class I CADs were expressed throughout development both in RNAseq data sets as well as via qRT-PCR analysis. Of the 37 class II TaCADs identified, two groups were observed to be significantly co-expressed with class I TaCADs in developing tissue and under chitin elicitation in RNAseq data sets. These co-expressed class II TaCADs were also found to be phylogenetically unrelated to a separate clade of class II TaCADs previously reported to be an influential resistance factor to pathogenic fungal infection. Lastly, two groups were phylogenetically identified as class III TaCADs, which possess distinct conserved gene structures. However, the lack of data supporting their catalytic activity for cinnamaldehydes and their bereft transcriptional presence in lignifying tissues challenges their designation and function as CADs. CONCLUSIONS: Taken together, our comprehensive transcriptomic analyses suggest that TaCAD genes contribute to overlapping but nonredundant functions during T. aestivum growth and development across a wide variety of agroecosystems and provide tolerance to various stressors.

Self-powered wearable biosensor based on stencil-printed carbon nanotube electrodes for ethanol detection in sweat.

Herein we introduce a novel water-based graphite ink modified with multiwalled carbon nanotubes, designed for the development of the first wearable self-powered biosensor enabling alcohol abuse detection through sweat analysis. The stencil-printed graphite (SPG) electrodes, printed onto a flexible substrate, were modified by casting multiwalled carbon nanotubes (MWCNTs), electrodepositing polymethylene blue (pMB) at the anode to serve as a catalyst for nicotinamide adenine dinucleotide (NADH) oxidation, and hemin at the cathode as a selective catalyst for H2O2 reduction. Notably, alcohol dehydrogenase (ADH) was additionally physisorbed onto the anodic electrode, and alcohol oxidase (AOx) onto the cathodic electrode. The self-powered biosensor was assembled using the ADH/pMB-MWCNTs/SPG||AOx/Hemin-MWCNTs/SPG configuration, enabling the detection of ethanol as an analytical target, both at the anodic and cathodic electrodes. Its performance was assessed by measuring polarization curves with gradually increasing ethanol concentrations ranging from 0 to 50 mM. The biosensor demonstrated a linear detection range from 0.01 to 0.3 mM, with a detection limit (LOD) of 3 ± 1 µM and a sensitivity of 64 ± 2 µW mM-1, with a correlation coefficient of 0.98 (RSD 8.1%, n = 10 electrode pairs). It exhibited robust operational stability (over 2800 s with continuous ethanol turnover) and excellent storage stability (approximately 93% of initial signal retained after 90 days). Finally, the biosensor array was integrated into a wristband and successfully evaluated for continuous alcohol abuse monitoring. This proposed system displays promising attributes for use as a flexible and wearable biosensor employing biocompatible water-based inks, offering potential applications in forensic contexts.

Retinol dehydrogenase 10 promotes epithelial-mesenchymal transition in spinal cord gliomas via PI3K/AKT pathway.

Background: Spinal cord glioma (SCG), a rare subset of central nervous system (CNS) glioma, represents a complex challenge in neuro-oncology. There has been research showing that Retinol Dehydrogenase 10 (RDH10) may be a tumor promoting factor in brain glioma, but the biological effects of RDH10 remain undefined in SCG. Methods: We performed gene set enrichment analysis (GSEA) and unsupervised clustering analysis to investigate the roles of EMT (epithelial-mesenchymal transition) in glioma. DEG (differently expressed gene) screening and correlation analysis were conducted to filter the candidate genes which were closely associated with EMT process in SCG. Enrichment analysis and GSVA (Gene Set Variation Analysis) were conducted to investigate the potential mechanism of RDH10 for SCG. Trans-well and healing assay were performed to explore the role of RDH10 in the invasion of SCG. Western blotting was performed to evaluate the levels of markers in PI3K-AKT and EMT pathway. In vivo tests were conducted to verify the role of RDH10 in EMT process. Results: Bioinformatic analysis demonstrated the EMT pathway was associated with dismal prognosis of glioma. Further analysis demonstrated that RDH10 showed the strongest correlation with the EMT process. Retinol Dehydrogenase 10 expression was significantly increased in SCG tissues, correlating with advanced tumor grade and unfavorable prognosis. Functional analysis indicated that decreasing RDH10 levels impeded the invasive and migratory abilities of SCG cells, whereas increasing RDH10 levels augmented them. Enrichment analysis and western blot revealed that RDH10 regulated EMT process of SCG by PI3K-AKT pathway. We observed that the enhanced invasion ability and increased EMT-related protein induced by RDH10 overexpression can be suppressed by PI3K-AKT pathway inhibitor (LY294002). Conclusion: Our research found that RDH10 was an effective biomarker associated with tumor grade and prognosis of SCG. RDH10 could regulate EMT process of SCG through PI3K-AKT pathway.

Efficient 2,3-Butanediol Production from Ethanol by a Modified Four-Enzyme Synthetic Biosystem.

2,3-butanediol (2,3-BD) is a versatile bio-based platform chemical. An artificial four-enzyme synthetic biosystem composed of ethanol dehydrogenase, NADH oxidase, formolase and 2,3-butanediol dehydrogenase was designed for upgrading ethanol to 2,3-BD in our previous study. However, a key challenge in developing in vitro enzymatic systems for 2,3-BD synthesis is the relatively sluggish catalytic efficiency of formolase, which catalyzes the rate-limiting step in such systems. Herein, this study reports how engineering the tunnel and substrate binding pocket of FLS improved its catalytic performance. A series of single-point and combinatorial variants were successfully obtained which displayed both higher catalytic efficiency and better substrate tolerance than wild-type FLS. Subsequently, a cell-free biosystem based on the FLS:I28V/L482E enzyme was implemented for upgrading ethanol to 2,3-BD. Ultimately, this system achieved efficient production of 2,3-BD from ethanol by the fed-batch method, reaching a concentration of 1.39 M (124.83 g/L) of the product and providing both excellent productivity and yield values of 5.94 g/L/h and 92.7%, respectively. Taken together, this modified enzymatic catalysis system provides a highly promising alternative approach for sustainable and cost-competitive production of 2,3-BD.

Report From the Second Global Scientific Conference on Clinical Trial Design and Outcome Measures for RDH12-Associated Inherited Retinal Degeneration.

Translational Relevance: A multi-stakeholder, patient centric approach will be critical to the design of future successful clinical trials with outcome measures relevant to the RDH12-IRD population.

Conserved amino acid residues and gene expression patterns associated with the substrate preferences of the competing enzymes FLS and DFR.

BACKGROUND: Flavonoids, an important class of specialized metabolites, are synthesized from phenylalanine and present in almost all plant species. Different branches of flavonoid biosynthesis lead to products like flavones, flavonols, anthocyanins, and proanthocyanidins. Dihydroflavonols form the branching point towards the production of non-colored flavonols via flavonol synthase (FLS) and colored anthocyanins via dihydroflavonol 4-reductase (DFR). Despite the wealth of publicly accessible data, there remains a gap in understanding the mechanisms that mitigate competition between FLS and DFR for the shared substrate, dihydroflavonols. RESULTS: An angiosperm-wide comparison of FLS and DFR sequences revealed the amino acids at positions associated with the substrate specificity in both enzymes. A global analysis of the phylogenetic distribution of these amino acid residues revealed that monocots generally possess FLS with Y132 (FLSY) and DFR with N133 (DFRN). In contrast, dicots generally possess FLSH and DFRN, DFRD, and DFRA. DFRA, which restricts substrate preference to dihydrokaempferol, previously believed to be unique to strawberry species, is found to be more widespread in angiosperms and has evolved independently multiple times. Generally, angiosperm FLS appears to prefer dihydrokaempferol, whereas DFR appears to favor dihydroquercetin or dihydromyricetin. Moreover, in the FLS-DFR competition, the dominance of one over the other is observed, with typically only one gene being expressed at any given time. CONCLUSION: This study illustrates how almost mutually exclusive gene expression and substrate-preference determining residues could mitigate competition between FLS and DFR, delineates the evolution of these enzymes, and provides insights into mechanisms directing the metabolic flux of the flavonoid biosynthesis, with potential implications for ornamental plants and molecular breeding strategies.

Development and Evaluation of Bis-benzothiazoles as a New Class of Benzothiazoles Targeting DprE1 as Antitubercular Agents.

Benzothiazole-bearing compounds have emerged as potential noncovalent DprE1 (decaprenylphosphoryl-ß-d-ribose-2'-epimerase) inhibitors active against Mycobacterium tuberculosis. Based on structure-based virtual screening (PDB ID: 4KW5), a focused library of thirty-one skeletally diverse benzothiazole amides was prepared, and the compounds were assessed for their antitubercular activity against M.tb H37Ra. Most potent compounds 3b and 3n were further evaluated against the M.tb H37Rv strain by the microdilution assay method. Among the compounds evaluated, bis-benzothiazole amide 3n emerged as a hit molecule and demonstrated promising antitubercular activity with minimum inhibitory concentration (MIC) values of 0.45 µg/mL and 8.0 µg/mL against H37Ra and H37Rv, respectively. Based on the preliminary hit molecule (3n), a focused library of 12 more bis-benzothiazole amide derivatives was further prepared by varying the substituents on either side to obtain new leads and generate a structure-activity relationship (SAR). Among these compounds, 6a, 6c, and 6d demonstrated remarkable antitubercular activity with MIC values of 0.5 µg/mL against H37Ra and 1.0, 2.0, and 8.0 µg/mL against H37Rv, respectively. The most active compound, 6a, also displayed significant efficacy against four drug-resistant tuberculosis strains. Compound 6a was assessed for in vitro cytotoxicity against the HepG2 cell line, and it displayed insignificant cytotoxicity. Furthermore, time-kill kinetic studies demonstrated time- and dose-dependent bactericidal activity of this compound. The GFP release assay revealed that compound 6a targets the inhibition of a cell wall component. SNPs in dprE-1 gene assessment revealed that compound 6a binds to tyrosine at position 314 of DprE1 and replaces it with histidine, causing resistance similar to that of standard TCA1. In silico docking studies further suggest that the strong noncovalent interactions of these compounds may lead to the development of potent noncovalent DprE1 inhibitors.

A new UiO-66-NH2 MOF-based nano-immobilized DFR enzyme as a biocatalyst for the synthesis of anthocyanidins.

Anthocyanidins and anthocyanins are one subclass of flavonoids in plants with diverse biological functions and have health-promoting effects. Dihydroflavonol 4-reductase (DFR) is one of the important enzymes involved in the biosynthesis of anthocyanidins and other flavonoids. Here, a new MOF-based nano-immobilized DFR enzyme acting as a nano-biocatalyst for the production of anthocyanidins in vitro was designed. We prepared UiO-66-NH2 MOF nano-carrier and recombinant DFR enzyme from genetic engineering. DFR@UiO-66-NH2 nano-immobilized enzyme was constructed based on covalent bonding under the optimum immobilization conditions of the enzyme/carrier ratio of 250 mg/g, 37 °C, pH 6.5 and fixation time of 10 min. DFR@UiO-66-NH2 was characterized and its catalytic function for the synthesis of anthocyanidins in vitro was testified using UPLC-QQQ-MS analysis. Compared with free DFR enzyme, the enzymatic reaction catalyzed by DFR@UiO-66-NH2 was more easily for manipulation in a wide range of reaction temperatures and pH values. DFR@UiO-66-NH2 had better thermal stability, enhanced adaptability, longer-term storage, outstanding tolerances to the influences of several organic reagents and Zn2+, Cu2+ and Fe2+ ions, and relatively good reusability. This work developed a new MOF-based nano-immobilized biocatalyst that had a good prospect of application in the green synthesis of anthocyanins in the future.

Formulation and clinical evaluation of carbidopa/levodopa oral solution for the treatment of sepiapterin reductase deficiency.

BACKGROUND: sepiapterine reductase deficiency (SRD) is a rare levodopa (L-dopa)-responsive disorder treated with a combination therapy of controlled-release L-dopa and carbidopa. The currently available formulation of controlled-release carbidopa/L-dopa does not entirely meet the requirements for the long-term therapy in pediatric patients. In fact, administration of a manufactured tablet at a dose intended for adults necessitates its adjustment to the child's needs, as the splitting of the tablet into smaller portions or its dilution in water. It's essential to emphasize that tablets must not be crushed, as this can compromise the controlled-release mechanism and affect the efficacy of the medication. At the moment, commercial liquid formulations are not available. Given these limitations, in house drug preparation in hospitals and community pharmacies is a valid option to ensure the proper therapeutic management of these patients. MATERIALS AND METHODS: we described sample preparation, physical and microbiological analyses, taste testing, and tolerability of a 1:10 ratio carbidopa/L-dopa flavored (mint, raspberry, cacao, berries) and unflavored oral formulation (no sweetening agents were added). We also reported long-term follow-up of two pediatric patients with SRD. RESULTS: we documented the stability for 28 days at 25 °C of the liquid solution. All formulations were well-tolerated, and no adverse events were observed during or after assessing taste and tolerability. The long-term follow up of two patients was characterized by effective symptom control and optimal treatment adherence and compliance. CONCLUSIONS: in-house liquid drug formulations can be a valid option for pediatric patients with SRD. Given the significant impact of taste on medication adherence, the use of flavoring agents in the development of liquid formulations of L-dopa/carbidopa results a very useful strategy to obtain optimal adherence in the pediatric population.

Design, construction and optimization of formaldehyde growth biosensors with broad application in biotechnology.

Formaldehyde is a key metabolite in natural and synthetic one-carbon metabolism. To facilitate the engineering of formaldehyde-producing enzymes, the development of sensitive, user-friendly, and cost-effective detection methods is required. In this study, we engineered Escherichia coli to serve as a cellular biosensor capable of detecting a broad range of formaldehyde concentrations. Using both natural and promiscuous formaldehyde assimilation enzymes, we designed three distinct E. coli growth biosensor strains that depend on formaldehyde for cell growth. These strains were engineered to be auxotrophic for one or several essential metabolites that could be produced through formaldehyde assimilation. The respective assimilating enzyme was expressed from the genome to compensate the auxotrophy in the presence of formaldehyde. We first predicted the formaldehyde dependency of the biosensors by flux balance analysis and then analysed it experimentally. Subsequent to strain engineering, we enhanced the formaldehyde sensitivity of two biosensors either through adaptive laboratory evolution or modifications at metabolic branch points. The final set of biosensors demonstrated the ability to detect formaldehyde concentrations ranging approximately from 30 µM to 13 mM. We demonstrated the application of the biosensors by assaying the in vivo activity of different methanol dehydrogenases in the most sensitive strain. The fully genomic nature of the biosensors allows them to be deployed as "plug-and-play" devices for high-throughput screenings of extensive enzyme libraries. The formaldehyde growth biosensors developed in this study hold significant promise for advancing the field of enzyme engineering, thereby supporting the establishment of a sustainable one-carbon bioeconomy.

Metabolic engineering of Komagataella phaffii for the efficient utilization of methanol.

BACKGROUND: Komagataella phaffii, a type of methanotrophic yeast, can use methanol, a favorable non-sugar substrate in eco-friendly bio-manufacturing. The dissimilation pathway in K. phaffii leads to the loss of carbon atoms in the form of CO2. However, the ΔFLD strain, engineered to lack formaldehyde dehydrogenase-an essential enzyme in the dissimilation pathway-displayed growth defects when exposed to a methanol-containing medium. RESULTS: Inhibiting the dissimilation pathway triggers an excessive accumulation of formaldehyde and a decline in the intracellular NAD+/NADH ratio. Here, we designed dual-enzyme complex with the alcohol oxidase1/dihydroxyacetone synthase1 (Aox1/Das1), enhancing the regeneration of the formaldehyde receptor xylulose-5-phosphate (Xu5P). This strategy mitigated the harmful effects of formaldehyde accumulation and associated toxicity to cells. Concurrently, we elevated the NAD+/NADH ratio by overexpressing isocitrate dehydrogenase in the TCA cycle, promoting intracellular redox homeostasis. The OD600 of the optimized combination of the above strategies, strain DF02-1, was 4.28 times higher than that of the control strain DF00 (ΔFLD, HIS4+) under 1% methanol. Subsequently, the heterologous expression of methanol oxidase Mox from Hansenula polymorpha in strain DF02-1 resulted in the recombinant strain DF02-4, which displayed a growth at an OD600 4.08 times higher than that the control strain DF00 in medium containing 3% methanol. CONCLUSIONS: The reduction of formaldehyde accumulation, the increase of NAD+/NADH ratio, and the enhancement of methanol oxidation effectively improved the efficient utilization of a high methanol concentration by strain ΔFLD strain lacking formaldehyde dehydrogenase. The modification strategies implemented in this study collectively serve as a foundational framework for advancing the efficient utilization of methanol in K. phaffii.

A pH-dependent shift of redox cofactor specificity in a benzyl alcohol dehydrogenase of aromatoleum aromaticum EbN1.

We characterise a reversible bacterial zinc-containing benzyl alcohol dehydrogenase (BaDH) accepting either NAD+ or NADP+ as a redox cofactor. Remarkably, its redox cofactor specificity is pH-dependent with the phosphorylated cofactors favored at lower and the dephospho-forms at higher pH. BaDH also shows different steady-state kinetic behavior with the two cofactor forms. From a structural model, the pH-dependent shift may affect the charge of a histidine in the 2'-phosphate-binding pocket of the redox cofactor binding site. The enzyme is phylogenetically affiliated to a new subbranch of the Zn-containing alcohol dehydrogenases, which share this conserved residue. BaDH appears to have some specificity for its substrate, but also turns over many substituted benzyl alcohol and benzaldehyde variants, as well as compounds containing a conjugated C=C double bond with the aldehyde carbonyl group. However, compounds with an sp3-hybridised C next to the alcohol/aldehyde group are not or only weakly turned over. The enzyme appears to contain a Zn in its catalytic site and a mixture of Zn and Fe in its structural metal-binding site. Moreover, we demonstrate the use of BaDH in an enzyme cascade reaction with an acid-reducing tungsten enzyme to reduce benzoate to benzyl alcohol. KEY POINTS: •Zn-containing BaDH has activity with either NAD + or NADP+ at different pH optima. •BaDH converts a broad range of substrates. •BaDH is used in a cascade reaction for the reduction of benzoate to benzyl alcohol.

Focal dystonia in an adult with L-2- hydroxyglutaric aciduria.

L-2-Hydroxyglutaric aciduria (L-2-HGA) is a rare disorder. The patients have psychomotor retardation, ataxia, macrocephaly, and epilepsy usually in childhood. We present a case of L-2-HGA who developed dystonia in the third decade of life. The family reported symptoms of progressive psychomotor regression since childhood. On assessment, the patient had mild impairment of higher mental functions, mild exotropia, and right-hand dystonia. Brain MRI revealed diffuse bilateral symmetrical subcortical white matter hyperintense signals. 2-hydroxyglutaric acid in urine was elevated and the whole genome sequencing revealed a homogeneous pathogenic variant of the L-2-hydroxyglutarate dehydrogenase (L2HGDH) gene. The prognosis was explained to the caregivers. Patients with mild phenotype L-2-HGA can remain undiagnosed until adulthood. Cases of dystonia even without complaints of epilepsy should be investigated by MRI -brain, urine test and genetic testing to rule out L-2-HGA.

Catalytic Stability of S-1-(4-Hydroxyphenyl)-Ethanol Dehydrogenase from Aromatoleum aromaticum.

Derived from the denitrifying bacterium Aromatoleum aromaticum EbN1 (Azoarcus sp.), the enzyme S-1-(4-hydroxyphenyl)-ethanol dehydrogenase (S-HPED) belongs to the short-chain dehydrogenase/reductase family. Using research techniques like UV-Vis spectroscopy, dynamic light scattering, thermal-shift assay and HPLC, we investigated the catalytic and structural stability of S-HPED over a wide temperature range and within the pH range of 5.5 to 9.0 under storage and reaction conditions. The relationship between aggregation and inactivation of the enzyme in various pH environments was also examined and interpreted. At pH 9.0, where the enzyme exhibited no aggregation, we characterized thermally induced enzyme inactivation. Through isothermal and multitemperature analysis of inactivation data, we identified and confirmed the first-order inactivation mechanism under these pH conditions and determined the kinetic parameters of the inactivation process. Additionally, we report the positive impact of glucose as an enzyme stabilizer, which slows down the dynamics of S-HPED inactivation over a wide range of pH and temperature and limits enzyme aggregation. Besides characterizing the stability of S-HPED, the enzyme's catalytic activity and high stereospecificity for 10 prochiral carbonyl compounds were positively verified, thus expanding the spectrum of substrates reduced by S-HPED. Our research contributes to advancing knowledge about the biocatalytic potential of this catalyst.

Carrier-free immobilized enzymatic reactor based on CipA-fused carbonyl reductase for efficient synthesis of chiral alcohol with cofactor self-sufficiency.

In this study, based on the self-assembly strategy, we fused CipA with carbonyl reductase LXCARS154Y derived from Leifsonia xyli by gene coding, and successfully performed the carrier-free immobilization of LXCARS154Y. The immobilized enzyme was then characterized using scanning electron microscope (SEM), dynamic light scattering (DLS) and fourier transform infrared spectroscopy (FTIR). Compared with the free enzyme, the immobilized LXCARS154Y exhibited a 2.3-fold improvement in the catalytic efficiency kcat/km for the synthesis of a chiral pharmaceutical intermediate (R)-3,5-bis(trifluoromethyl)phenyl ethanol ((R)-BTPE) by reducing 3,5-bis(trifluoromethyl)acetophenone (BTAP). Moreover, the immobilized enzyme showed the enhanced stability while maintaining over 61 % relative activity after 18 cycles of batch reaction. Further, when CipA-fused carbonyl reductase was employed for (R)-BTPE production in a continuous flow reaction, almost complete yield (97.0 %) was achieved within 7 h at 2 M (512.3 g/L) of BTAP concentration, with a space-time yield of 1717.1 g·L-1·d-1. Notably, we observed the retention of cofactor NADH by CipA-based enzyme aggregates, resulting in a higher total turnover number (TTN) of 4815 to facilitate this bioreductive process. This research developed a concise strategy for efficient preparation of chiral intermediate with cofactor self-sufficiency via continuous flow biocatalysis, and the relevant mechanism was also explored.

Structure modeling-based characterization of ChnD, the 6-hydroxyhexanoate dehydrogenase from Acinetobacter sp. strain NCIMB 9871.

α,ω-Dicarboxylic acids, ω-aminoalkanoic acids, and α,ω-diaminoalkanes are valuable building blocks for the production of biopolyesters and biopolyamides. One of the key steps in producing these chemicals is the oxidation of ω-hydroxycarboxylic acids using alcohol dehydrogenases (e.g., ChnD of Acinetobacter sp. NCIMB 9871). However, the reaction and structural features of these enzymes remain mostly undiscovered. Thereby, we have investigated characteristics of ChnD based on enzyme kinetics, substrate-docking simulations, and mutation studies. Kinetic analysis revealed a distinct preference of ChnD for medium chain ω-hydroxycarboxylic acids, with the highest catalytic efficiency of 18.0 mM-1s-1 for 12-hydroxydodecanoic acid among C6 to C12 ω-hydroxycarboxylic acids. The high catalytic efficiency was attributed to the positive interactions between the carboxyl group of the substrates and the guanidino group of two arginine residues (i.e., Arg62 and Arg266) in the substrate binding site. The ChnD_R62L variant showed the increased efficiency and affinity, particularly for fatty alcohols (i.e., C6-C10) and branched-chain fatty alcohols, such as 3-methyl-2-buten-1-ol. Overall, this study contributes to the deeper understanding of medium-chain primary aliphatic alcohol dehydrogenases and their applications for the production of industrially relevant chemicals such as α,ω-dicarboxylic acids, ω-aminoalkanoic acids, and α,ω-diaminoalkanes from renewable biomass.

Kinetic characterization of the C-terminal domain of Malonyl-CoA reductase.

Malonyl-CoA reductase utilizes two equivalents of NADPH to catalyze the reduction of malonyl-CoA to 3-hydroxypropionic acid (3HP). This reaction is part of the carbon fixation pathway in the phototrophic bacterium Chloroflexus aurantiacus. The enzyme is composed of two domains. The C-terminal domain catalyzes the reduction of malonyl-CoA to malonic semialdehyde, while the N-terminal domain catalyzes the reduction of the aldehyde to 3HP. The two domains can be produced independently and retain their enzymatic activity. This report focuses on the kinetic characterization of the C-terminal domain. Initial velocity patterns and inhibition studies showed the kinetic mechanism is ordered with NADPH binding first followed by malonyl-CoA. Malonic semialdehyde is released first, while CoA and NADP+ are released randomly. Analogs of malonyl-CoA showed that the thioester carbon is reduced, while the carboxyl group is needed for proper positioning. The enzyme transfers the pro-S hydrogen of NADPH to malonyl-CoA and pH rate profiles revealed that a residue with a pKa value of about 8.8 must be protonated for activity. Kinetic isotope effects indicated that NADPH is not sticky (that is, NADPH dissociates from the enzyme faster than the rate of product formation) and product release is partially rate-limiting. Moreover, the mechanism is stepwise with the pH dependent step occurring before or after hydride transfer. The findings from this study will aid in the development of an eco-friendly biosynthesis of 3HP which is an industrial chemical used in the production of plastics and adhesives.