Structure of recombinant formate dehydrogenase from Methylobacterium extorquens (MeFDH1).

Formate dehydrogenase (FDH) is critical for the conversion between formate and carbon dioxide. Despite its importance, the structural complexity of FDH and difficulties in the production of the enzyme have made elucidating its unique physicochemical properties challenging. Here, we purified recombinant Methylobacterium extorquens AM1 FDH (MeFDH1) and used cryo-electron microscopy to determine its structure. We resolved a heterodimeric MeFDH1 structure at a resolution of 2.8 Å, showing a noncanonical active site and a well-embedded Fe-S redox chain relay. In particular, the tungsten bis-molybdopterin guanine dinucleotide active site showed an open configuration with a flexible C-terminal cap domain, suggesting structural and dynamic heterogeneity in the enzyme.

Tunnel engineering of gas-converting enzymes for inhibitor retardation and substrate acceleration.

Carbon monoxide dehydrogenase (CODH), formate dehydrogenase (FDH), hydrogenase (H2ase), and nitrogenase (N2ase) are crucial enzymatic catalysts that facilitate the conversion of industrially significant gases such as CO, CO2, H2, and N2. The tunnels in the gas-converting enzymes serve as conduits for these low molecular weight gases to access deeply buried catalytic sites. The identification of the substrate tunnels is imperative for comprehending the substrate selectivity mechanism underlying these gas-converting enzymes. This knowledge also holds substantial value for industrial applications, particularly in addressing the challenges associated with separation and utilization of byproduct gases. In this comprehensive review, we delve into the emerging field of tunnel engineering, presenting a range of approaches and analyses. Additionally, we propose methodologies for the systematic design of enzymes, with the ultimate goal of advancing protein engineering strategies.

Real flue gas CO2 hydrogenation to formate by an enzymatic reactor using O2- and CO-tolerant hydrogenase and formate dehydrogenase.

It is challenging to capture carbon dioxide (CO2), a major greenhouse gas in the atmosphere, due to its high chemical stability. One potential practical solution to eliminate CO2 is to convert CO2 into formate using hydrogen (H2) (CO2 hydrogenation), which can be accomplished with inexpensive hydrogen from sustainable sources. While industrial flue gas could provide an adequate source of hydrogen, a suitable catalyst is needed that can tolerate other gas components, such as carbon monoxide (CO) and oxygen (O2), potential inhibitors. Our proposed CO2 hydrogenation system uses the hydrogenase derived from Ralstonia eutropha H16 (ReSH) and formate dehydrogenase derived from Methylobacterium extorquens AM1 (MeFDH1). Both enzymes are tolerant to CO and O2, which are typical inhibitors of metalloenzymes found in flue gas. We have successfully demonstrated that combining ReSH- and MeFDH1-immobilized resins can convert H2 and CO2 in real flue gas to formate via a nicotinamide adenine dinucleotide-dependent cascade reaction. We anticipated that this enzyme system would enable the utilization of diverse H2 and CO2 sources, including waste gases, biomass, and gasified plastics.

Microbial engineering of Methylorubrum extorquens AM1 to enhance CO2 conversion into formate.

Methylorubrum extorquens AM1 has the potential to consume C1 feedstock to produce a wide range of biomaterials, from bioplastic to pharmaceutical. However, the synthetic biology tools for engineering M. extorquens AM1 need to be employed for precise control of recombinant enzyme expression. In this study, we presented an approach to improve the expression level of formate dehydrogenase 1 from M. extorquens AM1 (MeFDH1) using an efficient terminator and 5'-untranslated region (5'-UTR) design for enhanced carbon dioxide (CO2) conversion activity of whole-cell biocatalyst. The rrnB terminator significantly increased mRNA levels of MeFDH1 alpha and beta subunits by 8.2-fold and 11-fold, respectively, compared to the T7 terminator. Moreover, enzyme production was 1.6-fold higher with 2.1 mg/wet cell weight (WCW) using rrnB terminator. Homologous 5'-untranslated regions (5'-UTR) determined based on proteomics data and UTR designer also influenced the expression level of MeFDH1. The 5'-UTR of the formaldehyde activating enzyme (fae) was the strongest, with 2.5-fold higher expression than that of the control sequence (T7g-10L). Furthermore, the electrochemical reaction of recombinant strains as whole-cell biocatalysts was investigated for their applicability to CO2 conversion, showing enhanced formate productivity. The recombinant strain containing the 5'-UTR sequence of fae exhibited formate productivity of 5.0 mM/h, 2.3-fold higher than that of the control strain (T7). Overall, this study suggested practical applications for CO2 conversion into bioavailable formate and provided valuable insights for recombinant expression systems in methylotrophic strains.

Bioelectrochemical conversion of CO2 to value added product formate using engineered Methylobacterium extorquens.

The conversion of carbon dioxide to formate is a fundamental step for building C1 chemical platforms. Methylobacterium extorquens AM1 was reported to show remarkable activity converting carbon dioxide into formate. Formate dehydrogenase 1 from M. extorquens AM1 (MeFDH1) was verified as the key responsible enzyme for the conversion of carbon dioxide to formate in this study. Using a 2% methanol concentration for induction, microbial harboring the recombinant MeFDH1 expressing plasmid produced the highest concentration of formate (26.6 mM within 21 hours) in electrochemical reactor. 60 µM of sodium tungstate in the culture medium was optimal for the expression of recombinant MeFDH1 and production of formate (25.7 mM within 21 hours). The recombinant MeFDH1 expressing cells showed maximum formate productivity of 2.53 mM/g-wet cell/hr, which was 2.5 times greater than that of wild type. Thus, M. extorquens AM1 was successfully engineered by expressing MeFDH1 as recombinant enzyme to elevate the production of formate from CO2 after elucidating key responsible enzyme for the conversion of CO2 to formate.

Structural understanding of the recycling of oxidized ascorbate by dehydroascorbate reductase (OsDHAR) from Oryza sativa L. japonica.

Dehydroascorbate reductase (DHAR) is a key enzyme involved in the recycling of ascorbate, which catalyses the glutathione (GSH)-dependent reduction of oxidized ascorbate (dehydroascorbate, DHA). As a result, DHAR regenerates a pool of reduced ascorbate and detoxifies reactive oxygen species (ROS). In previous experiments involving transgenic rice, we observed that overexpression of DHAR enhanced grain yield and biomass. Since the structure of DHAR is not available, the enzymatic mechanism is not well-understood and remains poorly characterized. To elucidate the molecular basis of DHAR catalysis, we determined the crystal structures of DHAR from Oryza sativa L. japonica (OsDHAR) in the native, ascorbate-bound, and GSH-bound forms and refined their resolutions to 1.9, 1.7, and 1.7 Å, respectively. These complex structures provide the first information regarding the location of the ascorbate and GSH binding sites and their interacting residues. The location of the ascorbate-binding site overlaps with the GSH-binding site, suggesting a ping-pong kinetic mechanism for electron transfer at the common Cys20 active site. Our structural information and mutagenesis data provide useful insights into the reaction mechanism of OsDHAR against ROS-induced oxidative stress in rice.

Cold adaptation of a psychrophilic chaperonin from Psychrobacter sp. and its application for heterologous protein expression.

OBJECTIVES: A chaperonin, PsyGroELS, from the Antarctic psychrophilic bacterium Psychrobacter sp. PAMC21119, was examined for its role in cold adaptation when expressed in a mesophilic Escherichia coli strain. RESULTS: Growth of E. coli harboring PsyGroELS at 10 °C was increased compared to the control strain. A co-expression system using PsyGroELS was developed to increase productivity of the psychrophilic enzyme PsyEst9. PsyEst9 was cloned and expressed using three E. coli variants that co-expressed GroELS from PAMC21119, E. coli, or Oleispira antarctica RB8(T). Co-expression with PsyGroELS was more effective for the production of PsyEst9 compared tothe other chaperonins. CONCLUSION: PsyGroELS confers cold tolerance to E. coli, and shows potential as an effective co-expression system for the stable production of psychrophilic proteins.

Lipase-catalyzed enantioselective synthesis of (R,R)-lactide from alkyl lactate to produce PDLA (poly D-lactic acid) and stereocomplex PLA (poly lactic acid).

R-lactide, a pivotal monomer for the production of poly (D-lactic acid) (PDLA) or stereocomplex poly (lactic acid) (PLA) was synthesized from alkyl (R)-lactate through a lipase-catalyzed reaction without racemization. From among several types of lipase, only lipase B from Candida antarctica (Novozym 435; CAL-B) was effective in the reaction that synthesized (R,R)-lactide. Enantiopure (R,R)-lactide, which consisted of over 99% enantiomeric excess, was synthesized from methyl (R)-lactate through CAL-B catalysis. Removal of the methanol by-product was critical to obtain a high level of lactide conversion. The (R,R)-lactide yield was 56% in a reaction containing 100 mg of Novozym 435, 10 mM methyl (R)-lactate and 1500 mg of molecular sieve 5A in methyl tert-butyl ether (MTBE). The important monomer (R,R)-lactide that is required for the production of the widely recognized bio-plastic PDLA and the PLA stereocomplex can be obtained using this novel synthetic method.

Enzymatic coproduction of biodiesel and glycerol carbonate from soybean oil and dimethyl carbonate.

The enzymatic coproduction of biodiesel and glycerol carbonate by the transesterification of soybean oil was studied using lipase as catalyst in organic solvent. To produce biodiesel and glycerol carbonate simultaneously, experiments were designed sequentially. Enzyme screening, the molar ratio of dimethyl carbonate (DMC) to soybean oil, reaction temperature and solvent effects were investigated. The results of enzyme screening, at 100 g/L Novozym 435 (immobilized Candida antarctica lipase B), biodiesel and glycerol carbonate showed conversions of 58.7% and 50.7%, respectively. The optimal conditions were 60 °C, 100 g/L Novozym 435, 6.0:1 molar ratio with tert-butanol as solvent: 84.9% biodiesel and 92.0% glycerol carbonate production was achieved.

LC-ESI/MS/MS method for rapid screening and confirmation of 44 exogenous anabolic steroids in human urine.

A sensitive and rapid method based on liquid chromatography-triple-quadrupole tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI) has been developed and validated for the screening and confirmation of 44 exogenous anabolic steroids (29 parent steroids and 15 metabolites) in human urine. The method involves an enzymatic hydrolysis, liquid-liquid extraction, and detection by LC-MS/MS. A triple-quadrupole mass spectrometer was operated in positive ESI mode with selected reaction monitoring (SRM) mode for the screening and product ion scan mode for the confirmation. The protonated molecular ions were used as precursor ions for the SRM analysis and product ion scan. The intraday and interday precisions of the target analytes at concentrations of the minimum required performance levels for the screening were 2-14% and 2-15%, respectively. The limits of detection for the screening and confirmation method were 0.1-10 ng/mL and 0.2-10 ng/mL, respectively, for 44 steroids. This method was successfully applied to analysis of urine samples from suspected anabolic steroid abusers.