A high catalytic efficiency and chemotolerant formate dehydrogenase from Bacillus simplex.

NAD+ -dependent formate dehydrogenase (FDH) catalyzes the conversion of formate and NAD+ to produce carbon dioxide and NADH. The reaction is biotechnologically important because FDH is widely used for NADH regeneration in various enzymatic syntheses. However, major drawbacks of this versatile enzyme in industrial applications are its low activity, requiring its utilization in large amounts to achieve optimal process conditions. Here, FDH from Bacillus simplex (BsFDH) was characterized for its biochemical and catalytic properties in comparison to FDH from Pseudomonas sp. 101 (PsFDH), a commonly used FDH in various biocatalytic reactions. The data revealed that BsFDH possesses high formate oxidizing activity with a kcat value of 15.3 ± 1.9 s-1 at 25°C compared to 7.7 ± 1.0 s-1 for PsFDH. At the optimum temperature (60°C), BsFDH exhibited 6-fold greater activity than PsFDH. The BsFDH displayed higher pH stability and a superior tolerance toward sodium azide and H2 O2 inactivation, showing a 200-fold higher Ki value for azide inhibition and remaining stable in the presence of 0.5% H2 O2 compared to PsFDH. The application of BsFDH as a cofactor regeneration system for the detoxification of 4-nitrophenol by the reaction of HadA, which produced a H2 O2 byproduct was demonstrated. The biocatalytic cascades using BsFDH demonstrated a distinct superior conversion activity because the system tolerated H2 O2 well. Altogether, the data showed that BsFDH is a robust enzyme suitable for future application in industrial biotechnology.

Structure and biochemical characterization of an extradiol 3,4-dihydroxyphenylacetate 2,3-dioxygenase from Acinetobacter baumannii.

3,4-Dihydroxyphenylacetate (DHPA) 2,3-dioxygenase (EC 1.13.11.15) from Acinetobacter baumannii (AbDHPAO) is an enzyme that catalyzes the 2,3-extradiol ring-cleavage of DHPA in the p-hydroxyphenylacetate (HPA) degradation pathway. While the biochemical reactions of various DHPAOs have been reported, only structures of DHPAO from Brevibacterium fuscum and their homologs are available. Here, we report the X-ray structure and biochemical characterization of an Fe2+-specific AbDHPAO that shares 12% sequence identity to the enzyme from B. fuscum. The 1.8 Å X-ray structure of apo-AbDHPAO was determined with four subunits per asymmetric unit, consistent with a homotetrameric structure. Interestingly, the αß-sandwiched fold of the AbDHPAO subunit is different from the dual ß-barrel-like motif of the well-characterized B. fuscum DHPAO structures; instead, it is similar to the structures of non-DHPA extradiol dioxygenases from Comamonas sp. and Sphingomonas paucimobilis. Similarly, these extradiol dioxygenases share the same chemistry owing to a conserved 2-His-1-carboxylate catalytic motif. Structure analysis and molecular docking suggested that the Fe2+ cofactor and substrate binding sites consist of the conserved residues His12, His57, and Glu238 forming a 2-His-1-carboxylate motif ligating to Fe2+ and DHPA bound with Fe2+ in an octahedral coordination. In addition to DHPA, AbDHPAO can also use other 3,4-dihydroxyphenylacetate derivatives with different aliphatic carboxylic acid substituents as substrates, albeit with low reactivity. Altogether, this report provides a better understanding of the structure and biochemical properties of AbDHPAO and its homologs, which is advancing further modification of DHPAO in future applications.

Detection of cellular metabolites by redox enzymatic cascades.

Detection of cellular metabolites that are disease biomarkers is important for human healthcare monitoring and assessing prognosis and therapeutic response. Accurate and rapid detection of microbial metabolites and pathway intermediates is also crucial for the process optimization required for development of bioconversion methods using metabolically engineered cells. Various redox enzymes can generate electrons that can be employed in enzyme-based biosensors and in the detection of cellular metabolites. These reactions can directly transform target compounds into various readout signals. By incorporating engineered enzymes into enzymatic cascades, the readout signals can be improved in terms of accuracy and sensitivity. This review critically discusses selected redox enzymatic and chemoenzymatic cascades currently employed for detection of human- and microbe-related cellular metabolites including, amino acids, d-glucose, inorganic ions (pyrophosphate, phosphate, and sulfate), nitro- and halogenated phenols, NAD(P)H, fatty acids, fatty aldehyde, alkane, short chain acids, and cellular metabolites.

Luciferin Synthesis and Pesticide Detection by Luminescence Enzymatic Cascades.

D-Luciferin (D-LH2 ), a substrate of firefly luciferase (Fluc), is important for a wide range of bioluminescence applications. This work reports a new and green method using enzymatic reactions (HELP, HadA Enzyme for Luciferin Preparation) to convert 19 phenolic derivatives to 8 D-LH2 analogues with ≈51 % yield. The method can synthesize the novel 5'-methyl-D-LH2 and 4',5'-dimethyl-D-LH2 , which have never been synthesized or found in nature. 5'-Methyl-D-LH2 emits brighter and longer wavelength light than the D-LH2 . Using HELP, we further developed LUMOS (Luminescence Measurement of Organophosphate and Derivatives) technology for in situ detection of organophosphate pesticides (OPs) including parathion, methyl parathion, EPN, profenofos, and fenitrothion by coupling the reactions of OPs hydrolase and Fluc. The LUMOS technology can detect these OPs at parts per trillion (ppt) levels. The method can directly detect OPs in food and biological samples without requiring sample pretreatment.

Catalytic and structural insights into a stereospecific and thermostable Class II aldolase HpaI from Acinetobacter baumannii.

Aldolases catalyze the reversible reactions of aldol condensation and cleavage and have strong potential for the synthesis of chiral compounds, widely used in pharmaceuticals. Here, we investigated a new Class II metal aldolase from the p-hydroxyphenylacetate degradation pathway in Acinetobacter baumannii, 4-hydroxy-2-keto-heptane-1,7-dioate aldolase (AbHpaI), which has various properties suitable for biocatalysis, including stereoselectivity/stereospecificity, broad aldehyde utilization, thermostability, and solvent tolerance. Notably, the use of Zn2+ by AbHpaI as a native cofactor is distinct from other enzymes in this class. AbHpaI can also use other metal ion (M2+) cofactors, except Ca2+, for catalysis. We found that Zn2+ yielded the highest enzyme complex thermostability (Tm of 87 °C) and solvent tolerance. All AbHpaI•M2+ complexes demonstrated preferential cleavage of (4R)-2-keto-3-deoxy-D-galactonate ((4R)-KDGal) over (4S)-2-keto-3-deoxy-D-gluconate ((4S)-KDGlu), with AbHpaI•Zn2+ displaying the highest R/S stereoselectivity ratio (sixfold higher than other M2+ cofactors). For the aldol condensation reaction, AbHpaI•M2+ only specifically forms (4R)-KDGal and not (4S)-KDGlu and preferentially catalyzes condensation rather than cleavage by ∼40-fold. Based on 11 X-ray structures of AbHpaI complexed with M2+ and ligands at 1.85 to 2.0 Å resolution, the data clearly indicate that the M2+ cofactors form an octahedral geometry with Glu151 and Asp177, pyruvate, and water molecules. Moreover, Arg72 in the Zn2+-bound form governs the stereoselectivity/stereospecificity of AbHpaI. X-ray structures also show that Ca2+ binds at the trimer interface via interaction with Asp51. Hence, we conclude that AbHpaI•Zn2+ is distinctive from its homologues in substrate stereospecificity, preference for aldol formation over cleavage, and protein robustness, and is attractive for biocatalytic applications.

A Chemo-Enzymatic Cascade for the Smart Detection of Nitro- and Halogenated Phenols.

The flavin-dependent monooxygenase, HadA, catalyzes the dehalogenation and denitration of the toxicants, nitro- and halogenated phenols, to benzoquinone. The HadA reaction can be applied in one-pot reactions towards the de novo synthesis of d-luciferin by coupling with d-Cys condensation. d-luciferin, a valuable chemical widely used in biomedical applications, can be used as a substrate for the reaction of firefly luciferase to generate bioluminescence. As nitro- and halogenated phenols are key indicators of human overexposure to pesticides and pesticide contamination, the technology provides a sensitive and convenient tool for improved biomedical and environmental detection at ppb sensitivity in biological samples without the requirement for any pre-treatment. This dual-pronged method combines the advantages of waste biodetoxification to produce a valuable chemical as well as a smart detection tool for environmental and biomedical detection.

Identification of a Hotspot Residue for Improving the Thermostability of a Flavin-Dependent Monooxygenase.

HadA is a flavin-dependent monooxygenase that can catalyze the denitration and dehalogenation of a wide variety of toxicants such as pesticides. Although these enzymatic reactions are useful for bioremediation or biocatalysis, the application of HadA for these purposes is not yet possible because of its low thermostability. In this work we have engineered HadA to be more thermostable through the use of structural, in silico, and rational approaches. The X-ray structure of HadA was solved to obtain a reliable three-dimensional protein model for further prediction of thermostable variants. In silico analysis by using two bioinformatic tools-FireProt and Disulfide by Design-suggested 102 variants that we then further refined by applying rational criteria including the location of a particular residue and its nearby interactions, as well as other biophysical parameters to narrow down the list to six candidates. The G513Y variant was found to be an optimal engineered candidate because it has significantly improved stability relative to the wild-type enzyme and equivalent activity. G513Y has an activity half-life 72 (50 °C) and 160 times (45 °C) longer than that of the wild-type enzyme. Coupled together with thermostable reactions of reduced flavin and NADH-regenerating systems, the G513Y variant can be used to catalyze denitration of 4nitrophenol at 45 °C. Structure/sequence alignments of HadA and its homologues indicate that several flavin-dependent monooxygenases also contain amino acid residues homologous to the G513 of HadA, hence opening up the possibility of applying this engineering approach to improving their thermostabilities as well. Molecular dynamics (MD) simulations confirmed that the improved thermostability of the G513Y variant was due to aromatic hydrocarbon interactions between Y513 and N359, L347, G348, and F349.

3,4-Dihydroxyphenylacetate 2,3-dioxygenase from Pseudomonas aeruginosa: An Fe(II)-containing enzyme with fast turnover.

3,4-dihydroxyphenylacetate (DHPA) dioxygenase (DHPAO) from Pseudomonas aeruginosa (PaDHPAO) was overexpressed in Escherichia coli and purified to homogeneity. As the enzyme lost activity over time, a protocol to reactivate and conserve PaDHPAO activity has been developed. Addition of Fe(II), DTT and ascorbic acid or ROS scavenging enzymes (catalase or superoxide dismutase) was required to preserve enzyme stability. Metal content and activity analyses indicated that PaDHPAO uses Fe(II) as a metal cofactor. NMR analysis of the reaction product indicated that PaDHPAO catalyzes the 2,3-extradiol ring-cleavage of DHPA to form 5-carboxymethyl-2-hydroxymuconate semialdehyde (CHMS) which has a molar absorptivity of 32.23 mM-1cm-1 at 380 nm and pH 7.5. Steady-state kinetics under air-saturated conditions at 25°C and pH 7.5 showed a Km for DHPA of 58 ± 8 µM and a kcat of 64 s-1, indicating that the turnover of PaDHPAO is relatively fast compared to other DHPAOs. The pH-rate profile of the PaDHPAO reaction shows a bell-shaped plot that exhibits a maximum activity at pH 7.5 with two pKa values of 6.5 ± 0.1 and 8.9 ± 0.1. Study of the effect of temperature on PaDHPAO activity indicated that the enzyme activity increases as temperature increases up to 55°C. The Arrhenius plot of ln(k'cat) versus the reciprocal of the absolute temperature shows two correlations with a transition temperature at 35°C. Two activation energy values (Ea) above and below the transition temperature were calculated as 42 and 14 kJ/mol, respectively. The data imply that the rate determining steps of the PaDHPAO reaction at temperatures above and below 35°C may be different. Sequence similarity network analysis indicated that PaDHPAO belongs to the enzyme clusters that are largely unexplored. As PaDHPAO has a high turnover number compared to most of the enzymes previously reported, understanding its biochemical and biophysical properties should be useful for future applications in biotechnology.

Kinetic mechanism of L-α-glycerophosphate oxidase from Mycoplasma pneumoniae.

L-α-glycerophosphate oxidase is an FAD-dependent enzyme that catalyzes the oxidation of L-α-glycerophosphate (Glp) by molecular oxygen to generate dihydroxyacetone phosphate (DHAP) and hydrogen peroxide (H2O2). The catalytic properties of recombinant His6-GlpO from Mycoplasma pneumoniae (His6-MpGlpO) were investigated through transient and steady-state kinetics and ligand binding studies. The results indicate that the reaction mechanism of His6-MpGlpO follows a ping-pong model. Double-mixing mode stopped-flow experiments show that, after flavin-mediated substrate oxidation, DHAP leaves rapidly prior to the oxygen reaction. The values determined for the individual rate constants and kcat (4.2 s(-1) at 4 °C), in addition to the finding that H2 O2 binds to the oxidized enzyme, suggest that H2O2 release is the rate-limiting step for the overall reaction. The results indicate that His6 -MpGlpO contains mixed populations of fast- and slow-reacting species. It is predominantly the fast-reacting species that participates in turnover. In contrast to other GlpO enzymes previously described, His6-MpGlpO is able to catalyze the reverse reaction of reduced enzyme and DHAP. This result may be explained by the standard reduction potential value of His6-MpGlpO (-167 ± 1 mV), which is lower than those of GlpO from other species. We found that D,L-glyceraldehyde 3-phosphate (GAP) may be used as a substrate in the His6-MpGlpO reaction, although it exhibited an approximately 100-fold lower kcat value in comparison with the reaction of Glp. These results also imply involvement of GlpO in glycolysis, as well as in lipid and glycerol metabolism. The kinetic models and distinctive properties of His6-MpGlpO reported here should be useful for future drug development against Mycoplasma pneumoniae infection.