Cyclic CMP and cyclic UMP mediate bacterial immunity against phages.

The cyclic pyrimidines 3',5'-cyclic cytidine monophosphate (cCMP) and 3',5'-cyclic uridine monophosphate (cUMP) have been reported in multiple organisms and cell types. As opposed to the cyclic nucleotides 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP), which are second messenger molecules with well-established regulatory roles across all domains of life, the biological role of cyclic pyrimidines has remained unclear. Here we report that cCMP and cUMP are second messengers functioning in bacterial immunity against viruses. We discovered a family of bacterial pyrimidine cyclase enzymes that specifically synthesize cCMP and cUMP following phage infection and demonstrate that these molecules activate immune effectors that execute an antiviral response. A crystal structure of a uridylate cyclase enzyme from this family explains the molecular mechanism of selectivity for pyrimidines as cyclization substrates. Defense systems encoding pyrimidine cyclases, denoted here Pycsar (pyrimidine cyclase system for antiphage resistance), are widespread in prokaryotes. Our results assign clear biological function to cCMP and cUMP as immunity signaling molecules in bacteria.

An overview on the recently discovered iota-carbonic anhydrases.

Carbonic anhydrases (CAs, EC 4.2.1.1) have been studied for decades and have been classified as a superfamily of enzymes which includes, up to date, eight gene families or classes indicated with the Greek letters α, ß, γ, δ, ζ, η, θ, ι. This versatile enzyme superfamily is involved in multiple physiological processes, catalysing a fundamental reaction for all living organisms, the reversible hydration of carbon dioxide to bicarbonate and a proton. Recently, the ι-CA (LCIP63) from the diatom Thalassiosira pseudonana and a bacterial ι-CA (BteCAι) identified in the genome of Burkholderia territorii were characterised. The recombinant BteCAι was observed to act as an excellent catalyst for the physiologic reaction. Very recently, the discovery of a novel ι-CAs (COG4337) in the eukaryotic microalga Bigelowiella natans and the cyanobacterium Anabaena sp. PCC7120 has brought to light an unexpected feature for this ancient superfamily: this ι-CAs was catalytically active without a metal ion cofactor, unlike the previous reported ι-CAs as well as all known CAs investigated so far. This review reports recent investigations on ι-CAs obtained in these last three years, highlighting their peculiar features, and hypothesising that possibly this new CA family shows catalytic activity without the need of metal ions.

A Flavoprotein Dioxygenase Steers Bacterial Tropone Biosynthesis via Coenzyme A-Ester Oxygenolysis and Ring Epoxidation.

Bacterial tropone natural products such as tropolone, tropodithietic acid, or the roseobacticides play crucial roles in various terrestrial and marine symbiotic interactions as virulence factors, antibiotics, algaecides, or quorum sensing signals. We now show that their poorly understood biosynthesis depends on a shunt product from aerobic CoA-dependent phenylacetic acid catabolism that is salvaged by the dedicated acyl-CoA dehydrogenase-like flavoenzyme TdaE. Further characterization of TdaE revealed an unanticipated complex catalysis, comprising substrate dehydrogenation, noncanonical CoA-ester oxygenolysis, and final ring epoxidation. The enzyme thereby functions as an archetypal flavoprotein dioxygenase that incorporates both oxygen atoms from O2 into the substrate, most likely involving flavin-N5-peroxide and flavin-N5-oxide species for consecutive CoA-ester cleavage and epoxidation, respectively. The subsequent spontaneous decarboxylation of the reactive enzyme product yields tropolone, which serves as a key virulence factor in rice panicle blight caused by pathogenic edaphic Burkholderia plantarii. Alternatively, the TdaE product is most likely converted to more complex sulfur-containing secondary metabolites such as tropodithietic acid from predominant marine Rhodobacteraceae (e.g., Phaeobacter inhibens).

Physiological Role of the Previously Unexplained Benzenetriol Dioxygenase Homolog in the Burkholderia sp. Strain SJ98 4-Nitrophenol Catabolism Pathway.

4-Nitrophenol, a priority pollutant, is degraded by Gram-positive and Gram-negative bacteria via 1,2,4-benzenetriol (BT) and hydroquinone (HQ), respectively. All enzymes involved in the two pathways have been functionally identified. So far, all Gram-negative 4-nitrophenol utilizers are from the genera Pseudomonas and Burkholderia. But it remains a mystery why pnpG, an apparently superfluous BT 1,2-dioxygenase-encoding gene, always coexists in the catabolic cluster (pnpABCDEF) encoding 4-nitrophenol degradation via HQ. Here, the physiological role of pnpG in Burkholderia sp. strain SJ98 was investigated. Deletion and complementation experiments established that pnpG is essential for strain SJ98 growing on 4-nitrocatechol rather than 4-nitrophenol. During 4-nitrophenol degradation by strain SJ98 and its two variants (pnpG deletion and complementation strains), 1,4-benzoquinone and HQ were detected, but neither 4-nitrocatechol nor BT was observed. When the above-mentioned three strains (the wild type and complementation strains with 2,2'-dipyridyl) were incubated with 4-nitrocatechol, BT was the only intermediate detected. The results established the physiological role of pnpG that encodes BT degradation in vivo. Biotransformation analyses showed that the pnpA-deleted strain was unable to degrade both 4-nitrophenol and 4-nitrocatechol. Thus, the previously characterized 4-nitrophenol monooxygenase PnpASJ98 is also essential for the conversion of 4-nitrocatechol to BT. Among 775 available complete genomes for Pseudomonas and Burkholderia, as many as 89 genomes were found to contain the putative pnpBCDEFG genes. The paucity of pnpA (3 in 775 genomes) implies that the extension of BT and HQ pathways enabling the degradation of 4-nitrophenol and 4-nitrocatechol is rarer, more recent, and likely due to the release of xenobiotic nitroaromatic compounds. IMPORTANCE An apparently superfluous gene (pnpG) encoding BT 1,2-dioxygenase is always found in the catabolic clusters involved in 4-nitrophenol degradation via HQ by Gram-negative bacteria. Our experiments reveal that pnpG is not essential for 4-nitrophenol degradation in Burkholderia sp. strain SJ98 but instead enables its degradation of 4-nitrocatechol via BT. The presence of pnpG genes broadens the range of growth substrates to include 4-nitrocatechol or BT, intermediates from the microbial degradation of many aromatic compounds in natural ecosystems. In addition, the existence of pnpCDEFG in 11.6% of the above-mentioned two genera suggests that the ability to degrade BT and HQ simultaneously is ancient. The extension of BT and HQ pathways including 4-nitrophenol degradation seems to be an adaptive evolution for responding to synthetic nitroaromatic compounds entering the environment since the industrial revolution.

Effect of amino acids and amines on the activity of the recombinant ι-carbonic anhydrase from the Gram-negative bacterium Burkholderia territorii.

We here report a study on the activation of the ι-class bacterial CA from Burkholderia territorii (BteCAι). This protein was recently characterised as a zinc-dependent enzyme that shows a significant catalytic activity (kcat 3.0 × 105 s-1) for the physiological reaction of CO2 hydration to bicarbonate and protons. Some amino acids and amines, among which some proteinogenic derivatives as well as histamine, dopamine and serotonin, showed efficient activating properties towards BteCAι, with activation constants in the range 3.9-13.3 µM. L-Phe, L-Asn, L-Glu, and some pyridyl-alkylamines, showed a weaker activating effect towards BteCAι, with KA values ranging between 18.4 µM and 45.6 µM. Nowadays, no information is available on active site architecture, metal ion coordination and catalytic mechanism of members of the ι-group of CAs, and this study represents another contribution towards a better understanding of this still uncharacterised class of enzymes.

Production of itaconic acid from alkali pretreated lignin by dynamic two stage bioconversion.

Expanding the portfolio of products that can be made from lignin will be critical to enabling a viable bio-based economy. Here, we engineer Pseudomonas putida for high-yield production of the tricarboxylic acid cycle-derived building block chemical, itaconic acid, from model aromatic compounds and aromatics derived from lignin. We develop a nitrogen starvation-detecting biosensor for dynamic two-stage bioproduction in which itaconic acid is produced during a non-growth associated production phase. Through the use of two distinct itaconic acid production pathways, the tuning of TCA cycle gene expression, deletion of competing pathways, and dynamic regulation, we achieve an overall maximum yield of 56% (mol/mol) and titer of 1.3 g/L from p-coumarate, and 1.4 g/L titer from monomeric aromatic compounds produced from alkali-treated lignin. This work illustrates a proof-of-principle that using dynamic metabolic control to reroute carbon after it enters central metabolism enables production of valuable chemicals from lignin at high yields by relieving the burden of constitutively expressing toxic heterologous pathways.

Biological characteristics and salt-tolerant plant growth-promoting effects of an ACC deaminase-producing Burkholderia pyrrocinia strain isolated from the tea rhizosphere.

Plant growth-promoting rhizobacteria that produce 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase can promote plant growth and enhance abiotic stress tolerance. In this study, Burkholderia pyrrocinia strain P10, with an ACC deaminase activity of 33.01-µmol/h/mg protein, was isolated from the tea rhizosphere and identified based on morphological, biochemical, and molecular characteristics. In addition to its ACC deaminase activity at pH 5.0-9.0 and in response to 5% NaCl and 20% polyethylene glycol, strain P10 can also solubilize phosphorus compounds, produce indole-3-acetic acid, and secrete siderophores. Pot experiments revealed that strain P10 can significantly enhance peanut seedling growth under saline conditions (100- and 170-mmol/L NaCl). Specifically, it increased the fresh weight and root length of plants by 90.12% and 79.22%, respectively, compared with high-salt stress. These results provide new insights into the biological characteristics of Burkholderia pyrrocinia, which may be useful as a bio-fertilizer.

Identification of the gene encoding 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid desulfhydrase in Burkholderia sp. HME13.

Here, we report the identification of the gene encoding a novel enzyme, 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid desulfhydrase, in Burkholderia sp. HME13. The enzyme converts 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid and H2O to 3-(2,5-dioxoimidazolidin-4-yl) propionic acid and H2S. Amino acid sequence analysis of the enzyme indicates that it belongs to the DUF917 protein family, which consists of proteins of unknown function.

Specific immobilization of lipase on functionalized 3D printing scaffolds via enhanced hydrophobic interaction for efficient resolution of racemic 1-indanol.

Lipase immobilization with hydrophobic interaction is of interesting exploration, and some functionalized groups on supports are special for activity increasing. To achieved a good performance of cost-effective immobilization on macro-supports for feasible usage and recycle, eco-friendly PLA-based 3D printing macro-scaffolds with fabrication was designed, and phenyl groups with different length of linkers and combined two kinds of groups were anchored for lipase YCJ01 binding with improving payload, the highest enzyme expression of 2227.5 U/g, activity recovery of 137.3%, and increasing specific activity of 815.9 U/mg were attained by using PLA@AMTS-C7-Ph/PLA@AMTS-C9-Ph scaffolds as carries. The immobilized lipase YCJ01 on bifunctionalized 3D printing scaffolds was further applied to the efficient resolution of racemic 1-indanol (267 mM) with high stereoselectivity using a binary solvent system. The immobilized lipase YCJ01 could control the over transesterification of (S)-1-indanol and exhibit good operational stability of repetitive usage for 9 cycles. This is beneficial to obtain the high enantiomerical pure product by feasible separation of immobilized biocatalyst without rigorous operation.

Highly selective synthesis of D-amino acids via stereoinversion of corresponding counterpart by an in vivo cascade cell factory.

BACKGROUND: D-Amino acids are increasingly used as building blocks to produce pharmaceuticals and fine chemicals. However, establishing a universal biocatalyst for the general synthesis of D-amino acids from cheap and readily available precursors with few by-products is challenging. In this study, we developed an efficient in vivo biocatalysis system for the synthesis of D-amino acids from L-amino acids by the co-expression of membrane-associated L-amino acid deaminase obtained from Proteus mirabilis (LAAD), meso-diaminopimelate dehydrogenases obtained from Symbiobacterium thermophilum (DAPDH), and formate dehydrogenase obtained from Burkholderia stabilis (FDH), in recombinant Escherichia coli. RESULTS: To generate the in vivo cascade system, three strategies were evaluated to regulate enzyme expression levels, including single-plasmid co-expression, double-plasmid co-expression, and double-plasmid MBP-fused co-expression. The double-plasmid MBP-fused co-expression strain Escherichia coli pET-21b-MBP-laad/pET-28a-dapdh-fdh, exhibiting high catalytic efficiency, was selected. Under optimal conditions, 75 mg/mL of E. coli pET-21b-MBP-laad/pET-28a-dapdh-fdh whole-cell biocatalyst asymmetrically catalyzed the stereoinversion of 150 mM L-Phe to D-Phe, with quantitative yields of over 99% ee in 24 h, by the addition of 15 mM NADP+ and 300 mM ammonium formate. In addition, the whole-cell biocatalyst was used to successfully stereoinvert a variety of aromatic and aliphatic L-amino acids to their corresponding D-amino acids. CONCLUSIONS: The newly constructed in vivo cascade biocatalysis system was effective for the highly selective synthesis of D-amino acids via stereoinversion.

Effect of Sulfonamides and Their Structurally Related Derivatives on the Activity of ι-Carbonic Anhydrase from Burkholderia territorii

Carbonic anhydrases (CAs) are essential metalloenzymes in nature, catalyzing the carbon dioxide reversible hydration into bicarbonate and proton. In humans, breathing and many other critical physiological processes depend on this enzymatic activity. The CA superfamily function and inhibition in pathogenic bacteria has recently been the object of significant advances, being demonstrated to affect microbial survival/virulence. Targeting bacterial CAs may thus be a valid alternative to expand the pharmacological arsenal against the emergence of widespread antibiotic resistance. Here, we report an extensive study on the inhibition profile of the recently discovered ι-CA class present in some bacteria, including Burkholderia territorii, namely BteCAι, using substituted benzene-sulfonamides and clinically licensed sulfonamide-, sulfamate- and sulfamide-type drugs. The BteCAι inhibition profile showed: (i) several benzene-sulfonamides with an inhibition constant lower than 100 nM; (ii) a different behavior with respect to other α, ß and γ-CAs; (iii) clinically used drugs having a micromolar affinity. This prototype study contributes to the initial recognition of compounds which efficiently and selectively inhibit a bacterial member of the ι-CA class, for which such a selective inhibition with respect to other protein isoforms present in the host is highly desired and may contribute to the development of novel antimicrobials.

Anion inhibition studies of the Zn(II)-bound ι-carbonic anhydrase from the Gram-negative bacterium Burkholderia territorii.

Burkholderia territorii, a Gram-negative bacterium, encodes for the ι-class carbonic anhydrase (CA, EC 4.2.1.1) BteCAι, which was recently characterised. It acts as a good catalyst for the hydration of CO2 to bicarbonate and protons, with a kcat value of 3.0 × 105 s-1 and kcat/KM value of 3.9 × 107 M-1 s-1. No inhibition data on this new class of enzymes are available to date. We report here an anion and small molecules inhibition study of BteCAι, which we prove to be a zinc(II)- and not manganese(II)-containing enzyme, as reported for diatom ι-CAs. The best inhibitors were sulphamic acid, stannate, phenylarsonic acid, phenylboronic acid and sulfamide (KI values of 6.2-94 µM), whereas diethyldithiocarbamate, tellurate, selenate, bicarbonate and cyanate were submillimolar inhibitors (KI values of 0.71-0.94 mM). The halides (except iodide), thiocyanate, nitrite, nitrate, carbonate, bisulphite, sulphate, hydrogensulfide, peroxydisulfate, selenocyanate, fluorosulfonate and trithiocarbonate showed KI values in the range of 3.1-9.3 mM.

Performing under pressure: esterification activity of dry fermented solids in subcritical and supercritical CO2.

OBJECTIVE: Lipases are often used in immobilized form, but commercial immobilized lipases are costly. An alternative is to produce lipases in solid-state fermentation, dry the solids and then use the "dry fermented solids" (DFS) directly. We produced DFS by growing Burkholderia contaminans on a mixture of sugarcane bagasse and sunflower seed meal and used the DFS to esterify oleic acid with ethanol in subcritical and supercritical CO2 at 40 °C. RESULTS: Compared to a control without CO2 at atmospheric pressure, subcritical CO2 at 30 bar improved esterification activity 1.2-fold. Higher pressures, including supercritical pressures up to 150 bar, reduced activity to less than 80% of the control. At 30 bar, the esterification activity was improved a further 1.8-fold with the addition of 9% water (i.e. 9 g water per 100 g oleic acid) to the reaction medium. CONCLUSION: A subcritical CO2 atmosphere, with the addition of a small amount of water, improved the esterification activity of DFS containing lipases of Burkholderia contaminans.

Bacterial triacylglycerol lipase is a potential cholesterol esterase: Identification of a key determinant for sterol-binding specificity.

Cholesterol esterase (Che) from Burkholderia stabilis (BsChe) is a homolog of well-characterized and industrially relevant bacterial triacylglycerol lipases (Lips). BsChe is a rare bacterial Lip enzyme that exhibits practical Che activity and is currently used in clinical applications to determine total serum cholesterol levels. To investigate the sterol specificity of BsChe, we determined the X-ray structure of BsChe. We discovered a local structural change in the active-site cleft, which might be related to substrate binding and product release. We also performed molecular docking studies by using the X-ray models of BsChe and cholesterol linoleate (CLL), the most favorable substrate for BsChe. The results showed that the sterol moieties of reasonable CLL docking poses localized to a specific active-site cleft surface formed by Leu266 and Ile287, which are unconserved among Burkholderia Lip homologs. Site-directed mutagenesis identified these residues as essential for the Che activity of BsChe, and Leu or Ile substitution conferred marked Che activity to Burkholderia Lips. In particular, Burkholderia cepacia and Burkholderia ubonensis Lips with the V266L/L287I double mutation exhibited ~50-fold and 500-fold higher Che activities than those of the wild-type enzymes, respectively. These results provide new insights into the substrate-binding mechanisms and selectivities of bacterial Lips.

Efficient Synthesis of New Fluorinated ß-Amino Acid Enantiomers through Lipase-Catalyzed Hydrolysis.

An efficient and novel enzymatic method has been developed for the synthesis of ß-fluorophenyl-substituted ß-amino acid enantiomers through lipase PSIM (Burkholderia cepasia) catalyzed hydrolysis of racemic ß-amino carboxylic ester hydrochloride salts 3a-e in iPr2O at 45 °C in the presence of Et3N and H2O. Adequate analytical methods were developed for the enantio-separation of racemic ß-amino carboxylic ester hydrochlorides 3a-e and ß-amino acids 2a-e. Preparative-scale resolutions furnished unreacted amino esters (R)-4a-e and product amino acids (S)-5a-e with excellent ee values (≥99%) and good chemical yields (>48%).

A Peptidoglycan Amidase Mutant of Burkholderia insecticola Adapts an L-form-like Shape in the Gut Symbiotic Organ of the Bean Bug Riptortus pedestris.

Bacterial cell shapes may be altered by the cell cycle, nutrient availability, environmental stress, and interactions with other organisms. The bean bug Riptortus pedestris possesses a symbiotic bacterium, Burkholderia insecticola, in its midgut crypts. This symbiont is a typical rod-shaped bacterium under in vitro culture conditions, but changes to a spherical shape inside the gut symbiotic organ of the host insect, suggesting the induction of morphological alterations in B. insecticola by host factors. The present study revealed that a deletion mutant of a peptidoglycan amidase gene (amiC), showing a filamentous chain form in vitro, adapted a swollen L-form-like cell shape in midgut crypts. Spatiotemporal observations of the ΔamiC mutant in midgut crypts revealed the induction of swollen cells, particularly prior to the molting of insects. To elucidate the mechanisms underlying in vivo-specific morphological alterations, the symbiont was cultured under 13 different conditions and its cell shape was examined. Swollen cells, similar to symbiont cells in midgut crypts, were induced when the mutant was treated with fosfomycin, an inhibitor of peptidoglycan precursor biosynthesis. Collectively, these results strongly suggest that the Burkholderia symbiont in midgut crypts is under the control of the host insect via a cell wall-attacking agent.

Cloning of a novel topramezone-resistant 4-hydroxyphenylpyruvate dioxygenase gene and improvement of its resistance through pressure acclimation.

Topramezone is a new 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor herbicide that is widely used on corn to control annual grass weeds and broadleaf weeds. Due to its broad-spectrum weed control capacity, improved activity, excellent crop selectivity, low mammalian toxicity and high environmental safety, topramezone is considered an ideal target herbicide for transgenic engineering of herbicide tolerance. In this study, a topramezone-resistant strain, Burkholderia sp. BW-1, was screened from soil, and a novel topramezone-resistant HPPD gene (Bkhppd) was cloned from this strain. Purified BkHPPD displayed relatively high HPPD activity and topramezone resistance with a half-maximal inhibitory concentration (IC50) of 572.0 nM. Two BkHPPD mutants designated as BkHPPDt31 and BkHPPDt76 were screened through pressure acclimation. BkHPPDt31 contained three amino acid substitutions (H65D, N160 T and N258S), whereas BkHPPDt76 contained four amino acid substitutions (H65D, N160 T, N258S and N343 T). The topramezone IC50 values of BkHPPDt31 and BkHPPDt76 were 1.1- and 2.3-fold higher, respectively, than that of wild-type BkHPPD. In addition, site-directed mutagenesis indicated that the increased resistance conferred by BkHPPDt31 resulted from the synergistic effects of the three site mutations rather than a single site mutation, and that substitution of asparagine 343 with threonine significantly decreased catalytic efficiency and affinity but increased topramezone resistance. In summary, this study provides a novel topramezone-resistant HPPD gene for the engineering of genetically modified herbicide-resistant crops and facilitates further elucidation of the resistance mechanism of BkHPPD and improvement of resistance through directed evolution.

Cautionary Tale of Using Tris(alkyl)phosphine Reducing Agents with NAD+-Dependent Enzymes.

Protein biochemistry protocols typically include disulfide bond reducing agents to guard against unwanted thiol oxidation and protein aggregation. Commonly used disulfide bond reducing agents include dithiothreitol, ß-mercaptoethanol, glutathione, and the tris(alkyl)phosphine compounds tris(2-carboxyethyl)phosphine (TCEP) and tris(3-hydroxypropyl)phosphine (THPP). While studying the catalytic activity of the NAD(P)H-dependent enzyme Δ1-pyrroline-5-carboxylate reductase, we unexpectedly observed a rapid non-enzymatic chemical reaction between NAD+ and the reducing agents TCEP and THPP. The product of the reaction exhibits a maximum ultraviolet absorbance peak at 334 nm and forms with an apparent association rate constant of 231-491 M-1 s-1. The reaction is reversible, and nuclear magnetic resonance characterization (1H, 13C, and 31P) of the product revealed a covalent adduct between the phosphorus of the tris(alkyl)phosphine reducing agent and the C4 atom of the nicotinamide ring of NAD+. We also report a 1.45 Å resolution crystal structure of short-chain dehydrogenase/reductase with the NADP+-TCEP reaction product bound in the cofactor binding site, which shows that the adduct can potentially inhibit enzymes. These findings serve to caution researchers when using TCEP or THPP in experimental protocols with NAD(P)+. Because NAD(P)+-dependent oxidoreductases are widespread in nature, our results may be broadly relevant.

The metal- and substrate-dependences of 2,4'-dihydroxyacetophenone dioxygenase.

While the enzyme, 2,4'-dihydroxyacetophenone dioxygenase (DAD), has been known for decades, very little has been characterized of the mechanism of the DAD-catalyzed oxidative cleavage of its reported substrate, 2,4'-dihydroxyacetophenone (DHA). The purpose of this study was to identify the active metal center and to characterize the substrate-dependence of the kinetics of the reaction to lay the foundation for deeper mechanistic investigation. To this, the DAD V1M mutant (bDAD) was overexpressed, purified, and reconstituted with various metal ions. Kinetic assays evaluating the activity of the reconstituted enzyme as well as the substrate- and product-dependences of the reaction kinetics were performed. The results from reconstitution of the apoprotein with a variety of metal ions support the requirement for an Fe3+ center for enzyme activity. Reaction rates showed simple saturation kinetics for DHA with values for kcat and KDHA of 2.4 s-1 and 0.7 µM, respectively, but no significant dependence on the concentration of O2. A low-level inhibition (KI = 1100 µM) by the 4HB product was observed. The results support a minimal kinetic model wherein DHA binds to resting ferric enzyme followed by rapid addition of O2 to yield an intermediate complex that irreversibly collapses to products.

Rational Engineering of Formate Dehydrogenase Substrate/Cofactor Affinity for Better Performance in NADPH Regeneration.

Formate dehydrogenases are critical tools for nicotinamide cofactor regeneration, but their limited catalytic efficiency (kcat/Km) is a major drawback. A formate dehydrogenase from Burkholderia stabilis 15516 (BstFDH) was the first native NADP+-dependent formate dehydrogenase reported and has the highest kcat/Km toward NADP+ (kcat/KmNADP+) compared with other FDHs that can utilize NADP+ as a hydrogen acceptor. However, the substrate and cofactor affinities of BstFDH are inferior to those of other FDHs, making its practical application difficult. Herein, we engineered recombinant BstFDH to enhance its HCOO- and NADP+ affinities. Based on sequence information analysis and homologous modeling results, I124, G146, S262, and A287 were found to affect the binding affinity for HCOO- and NADP+. By combining these mutations, we identified a BstFDH variant (G146M/A287G) that reduced KmNADP+ to 0.09 mM, with a concomitant decrease in KmHCOO-, and gave 1.6-fold higher kcat/KmNADP+ than the wild type (WT). Furthermore, BstFDH I124V/G146H/A287G, with the lowest KmHCOO- of 8.51 mM, showed a catalytic efficiency that was 2.3-fold higher than that of the wild type and a decreased KmNADP+ of 0.11 mM. These results are beneficial for improving the performance of NADP+-dependent formate dehydrogenase in the NADPH regeneration of various bioreductive reactions and provide a useful guide for engineering of the substrate and cofactor affinity of other enzymes.