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Succinic semialdehyde dehydrogenase (SSADH) catalyses the conversion of succinic semialdehyde into succinic acid and two electrons are transferred to NAD(P)

Cofactor imbalance obstructs the productivities of metabolically engineered cells. Herein, we employed a minimally perturbing system, xylose reductase and lactose (XR/lactose), to increase levels of a pool of sugar-phosphates which are connected to the biosynthesis of NAD(P)H, FAD, FMN and ATP inEscherichia coli. The XR/lactose system could increase the amounts of the precursors of these cofactors and was tested with three different metabolically engineered cell systems (fatty alcohol biosynthesis, bioluminescence light generation and alkane biosynthesis) with different cofactor demands. Productivities of these cells were increased 2-4-fold by the XR/lactose system. Untargeted metabolomic analysis revealed different metabolite patterns among these cells; demonstrating that only metabolites involved in relevant cofactor biosynthesis were altered. The results were also confirmed by transcriptomic analysis. Another sugar reducing system (glucose dehydrogenase, GDH) could also be used to increase fatty alcohol production but resulted in less yield enhancement than XR. This work demonstrates that the approach of increasing cellular sugar phosphates can be a generic tool to increasein vivocofactor generation upon cellular demand for synthetic biology.TeaserUse of sugar and sugar reductase to increase sugar phosphates for enhancingin situsynthesis of cofactors upon cellular demand for synthetic biology.

Bacterial luciferase (Lux) catalyzes a bioluminescence reaction by using long-chain aldehyde, reduced flavin and molecular oxygen as substrates. The reaction can be applied in reporter gene systems for biomolecular detection in both prokaryotic and eukaryotic organisms. Because reduced flavin is unstable under aerobic conditions, another enzyme, flavin reductase, is needed to supply reduced flavin to the Lux-catalyzed reaction. To create a minimized cascade for Lux that would have greater ease of use, a chemoenzymatic reaction with a biomimetic nicotinamide (BNAH) was used in place of the flavin reductase reaction in the Lux system. The results showed that the minimized cascade reaction can be applied to monitor bioluminescence of the Lux reporter in eukaryotic cells effectively, and that it can achieve higher efficiencies than the system with flavin reductase. This development is useful for future applications as high-throughput detection tools for drug screening applications.

D-Luciferin (D-LH

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 A 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.

Luciferase-based gene reporters generating bioluminescence signals are important tools for biomedical research. Amongst the luciferases, flavin-dependent enzymes use the most common, and thus most economical chemicals. However, their applications in mammalian cells are limited due to their low signals compared to other systems. Here, we constructed Flavin Luciferase for Mammalian Cell Expression (FLUX) by engineering luciferase from Vibrio campbellii (the most thermostable bacterial luciferase reported to date) and optimizing its expression and reporter assays in mammalian cells. We found that the FLUX reporter gene can be overexpressed in various cell lines and showed outstanding signal-to-background in HepG2 cells, significantly higher than that of firefly luciferase (Fluc). The combined use of FLUX/Fluc as target/control vectors gave the most stable signals, better than the standard set of Fluc(target)/Rluc(control). We demonstrated that FLUX can be used for testing inhibitors of the NF-κB signaling pathway, validating FLUX applications for various assays in the future.

Reporter gene assays are powerful tools for monitoring dynamic molecular changes and for evaluating the responses that occur at the genetic elements within cells in response to exogenous molecules. In general, various protein systems can be used as reporter genes, including luciferases. Here, the present protocol introduces a unique reporter gene system for monitoring molecular events in cells using bacterial luciferase (lux), which can generate blue-green light suitable for gene reporter applications with the highest cost performance. The protocol also guides the assay conditions and necessary components for using of lux gene (lux) as a eukaryotic reporter system. The lux system can be applied to monitor variety of molecular events inside mammalian cellular systems.

Reporter gene assays are powerful tools for monitoring dynamic molecular changes and for evaluating the responses that occur at the genetic elements within cells in response to exogenous molecules. In general, various protein systems can be used as reporter genes, including luciferases. Here, the present protocol introduces a unique reporter gene system for monitoring molecular events in cells using bacterial luciferase (lux), which can generate blue-green light suitable for gene reporter applications with the highest cost performance. The protocol also guides the assay conditions and necessary components for using of lux gene (lux) as a eukaryotic reporter system. The lux system can be applied to monitor variety of molecular events inside mammalian cellular systems.

Bacterial luciferase is a flavin-dependent monooxygenase which is remarkable for its distinctive feature in transforming chemical energy to photons of visible light. The bacterial luciferase catalyzes bioluminescent reaction using reduced flavin mononucleotide, long-chain aldehyde and oxygen to yield oxidized flavin, corresponding acid, water and light at λ

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.

Succinic semialdehyde dehydrogenase (SSADH) from Acinetobacter baumannii (Ab) catalyzes the oxidation of succinic semialdehyde (SSA). This enzyme has two conserved cysteines (Cys289 and Cys291) and preferentially uses NADP+ over NAD+ as a hydride acceptor. Steady-state kinetic analysis showed that AbSSADH has the highest catalytic turnover (137 s-1 ) and can tolerate SSA inhibition the most (< 500 μm) among all SSADHs reported. Alanine substitutions of the two conserved cysteines indicated that Cys291Ala has ~ 65% activity compared with the wild-type enzyme while Cys289Ala is inactive, suggesting that Cys289 is the active residue participating in catalysis. Pre-steady-state kinetics showed for the first time burst kinetics for NADPH formation in SSADH, indicating that the rate-limiting step is associated with steps that occur after the hydride transfer. As the magnitude of burst kinetics represents the amount of NADPH formed during the first turnover, it is directly dependent on the amount of the deprotonated form of cysteine. The pKa of Cys289 was calculated from a plot of the burst magnitude vs pH as 7.4 ± 0.2. The Cys289 pKa was also measured based on the ability of AbSSADH to form an NADP-cysteine adduct, which can be detected by the increase of absorbance at ~ 330 nm as 7.9 ± 0.2. The lowering of the catalytic cysteine pKa by 0.6-1 unit renders the catalytic thiol more nucleophilic, which facilitates AbSSADH catalysis under physiological conditions. The methods established herein can specifically measure the active site cysteine pKa without interference from other cysteines. These techniques may be useful for studying ionization state of other cysteine-containing aldehyde dehydrogenases. ENZYME Succinic semialdehyde dehydrogenase, EC1.2.1.24.

Bacterial luciferase is a flavin-dependent monooxygenase which is remarkable for its distinctive feature in transforming chemical energy to photons of visible light. The bacterial luciferase catalyzes bioluminescent reaction using reduced flavin mononucleotide, long-chain aldehyde and oxygen to yield oxidized flavin, corresponding acid, water and light at λmax around 490nm. The enzyme comprises of two non-identical α and β subunits, where α subunit is a catalytic center and β subunit is crucially required for maintaining catalytic function of the α subunit. The crystal structure with FMN bound and mutagenesis studies have assigned a number of amino acid residues that are important in coordinating critical reactions and stabilizing intermediates to attain optimum reaction efficiency. The enzyme achieves monooxygenation by generating C4a-hydroperoxyflavin intermediate that later changes its protonation status to become C4a-peroxyflavin, which is necessary for the nucleophilic attacking with aldehyde substrate. The decomposing of C4a-peroxyhemiacetal produces excited C4a-hydroxyflavin and acid product. The chemical basis regrading bioluminophore generation in Lux reaction remains an inconclusive issue. However, current data can, at least, demonstrate the involvement of electron transfer to create radical molecules which is the key step in this mechanism. Lux is a self-sufficient bioluminescent system in which all substrates can be recycled and produced by a group of enzymes from the lux operon. This makes Lux distinctively advantageous over other luciferases for reporter enzyme application. The progression of understanding of Lux catalysis is beneficial to improve light emitting efficiency in order to expand the robustness of Lux application.

p-Hydroxyphenylacetate (HPA) can be derived from the biodegradation of lignin or from man-made compounds. The pathway involved for HPA degradation has been characterized for several species, but little is known on the degradation of HPA in Acinetobacter sp. In this report, the HPA degradation operon in A. baumannii TH was investigated using genome walking and PCR amplification to identify the genes encoded by the operon. The results showed that there are thirteen ORFs that are involved in this process and their arrangement in the operon of A. baumannii TH is different from that in the operons of other previously reported species. ORFs 8-12 show clear variation compared to orthologous genes from other species, particularly at ORF9 which encodes for succinic semialdehyde dehydrogenase (SSADH) that is absent in other species. The ssadh gene was overexpressed and the results confirmed that this enzyme is indeed succinate semialdehyde dehydrogenase. The results suggest that the final metabolites in this pathway are pyruvate and succinate, different from other species which have pyruvate and succinic semialdehyde as final products. Functional studies of the proteins encoded by ORF 8 and 10-12 have confirmed their roles in the HPA degradation pathway as an aldolase, a transporter protein, a hydroxylase and a reductase. Analysis of the sequence similarity network of enzymes encoded by ORFs 8-12 has revealed several interesting features. The designation of enzymes homologous to the oxygenase component of p-hydroxyphenylacetate 3-hydroxylase in the database should be reassigned, as they were mostly incorrectly assigned as acyl-CoA dehydrogenases. An understanding of the enzymatic reactions which convert aromatic compounds into pyruvate and succinate should be highly useful for future metabolic engineering for converting waste-derived aromatic compounds into useful biochemicals.

Succinic semialdehyde dehydrogenase (SSADH) from Acinetobacter baumannii (Ab) catalyzes the oxidation of succinic semialdehyde (SSA). This enzyme has two conserved cysteines (Cys289 and Cys291) and preferentially uses NADPSuccinic semialdehyde dehydrogenase, EC1.2.1.24.