Autoluminescent Plants

Prospects of obtaining plants glowing in the dark have captivated the imagination of scientists and layman alike. While light emission has been developed into a useful marker of gene expression, bioluminescence in plants remained dependent on externally supplied substrate. Evolutionary conservation of the prokaryotic gene expression machinery enabled expression of the six genes of the lux operon in chloroplasts yielding plants that are capable of autonomous light emission. This work demonstrates that complex metabolic pathways of prokaryotes can be reconstructed and function in plant chloroplasts and that transplastomic plants can emit light that is visible by naked eye

Structure of nitrilotriacetate monooxygenase component B from Mycobacterium thermoresistibile

The 1.6 Å resolution crystal structure of nitrilotriacetate monooxygenase component B (NTA-MoB) from M. thermoresistibile is presented, revealing a highly conserved C-terminal tail that may modulate the activity of NTA-MoB in mycobacteria

LuxG Is a Functioning Flavin Reductase for Bacterial Luminescence▿

The luxG gene is part of the lux operon of marine luminous bacteria. luxG has been proposed to be a flavin reductase that supplies reduced flavin mononucleotide (FMN) for bacterial luminescence. However, this role has never been established because the gene product has not been successfully expressed and characterized. In this study, luxG from Photobacterium leiognathi TH1 was cloned and expressed in Escherichia coli in both native and C-terminal His6-tagged forms. Sequence analysis indicates that the protein consists of 237 amino acids, corresponding to a subunit molecular mass of 26.3 kDa. Both expressed forms of LuxG were purified to homogeneity, and their biochemical properties were characterized. Purified LuxG is homodimeric and has no bound prosthetic group. The enzyme can catalyze oxidation of NADH in the presence of free flavin, indicating that it can function as a flavin reductase in luminous bacteria. NADPH can also be used as a reducing substrate for the LuxG reaction, but with much less efficiency than NADH. With NADH and FMN as substrates, a Lineweaver-Burk plot revealed a series of convergent lines characteristic of a ternary-complex kinetic model. From steady-state kinetics data at 4°C pH 8.0, Km for NADH, Km for FMN, and kcat were calculated to be 15.1 μM, 2.7 μM, and 1.7 s−1, respectively. Coupled assays between LuxG and luciferases from P. leiognathi TH1 and Vibrio campbellii also showed that LuxG could supply FMNH− for light emission in vitro. A luxG gene knockout mutant of P. leiognathi TH1 exhibited a much dimmer luminescent phenotype compared to the native P. leiognathi TH1, implying that LuxG is the most significant source of FMNH− for the luminescence reaction in vivo

LuxG Is a Functioning Flavin Reductase for Bacterial Luminescence

The 'luxG' gene is part of the 'lux' operon of marine luminous bacteria. 'luxG' has been proposed to be a flavin reductase that supplies reduced flavin mononucleotide (FMN) for bacterial luminescence. However, this role has never been established because the gene product has not been successfully expressed and characterized. In this study, 'luxG' from 'Photobacterium leiognathi' TH1 was cloned and expressed in 'Escherichia coli' in both native and C-terminal His6-tagged forms. Sequence analysis indicates that the protein consists of 237 amino acids, corresponding to a subunit molecular mass of 26.3 kDa. Both expressed forms of 'LuxG' were purified to homogeneity, and their biochemical properties were characterized. Purified 'LuxG' is homodimeric and has no bound prosthetic group. The enzyme can catalyze oxidation of NADH in the presence of free flavin, indicating that it can function as a flavin reductase in luminous bacteria. NADPH can also be used as a reducing substrate for the 'LuxG' reaction, but with much less efficiency than NADH. With NADH and FMN as substrates, a Lineweaver-Burk plot revealed a series of convergent lines characteristic of a ternary-complex kinetic model. From steady-state kinetics data at 4°C pH 8.0, Km for NADH, Km for FMN, and kcat were calculated to be 15.1 μM, 2.7 μM, and 1.7 s‾¹, respectively. Coupled assays between LuxG and luciferases from 'P. leiognathi' TH1 and Vibrio campbellii also showed that LuxG could supply FMNH‾ for light emission in vitro. A 'luxG' gene knockout mutant of 'P. leiognathi' TH1 exhibited a much dimmer luminescent phenotype compared to the native 'P. leiognathi' TH1, implying that 'LuxG' is the most significant source of FMNH‾ for the luminescence reaction in vivo

The Transfer of Reduced Flavin Mononucleotide from LuxG Oxidoreductase to Luciferase Occurs via Free Diffusion

Bacterial luciferase (LuxAB) is a two-component flavin mononucleotide (FMN)-dependent monooxygenase that catalyzes the oxidation of reduced FMN (FMNH^–) and a long-chain aliphatic aldehyde by molecular oxygen to generate oxidized FMN, the corresponding aliphatic carboxylic acid, and concomitant emission of light. The LuxAB reaction requires a flavin reductase to generate FMNH^– to serve as a luciferin in its reaction. However, FMNH^– is unstable and can react with oxygen to generate H₂O₂, so that it is important to transfer it efficiently to LuxAB. Recently, LuxG has been identified as a NADH:FMN oxidoreductase that supplies FMNH^– to luciferase <i>in vivo</i>. In this report, the mode of transfer of FMNH^– between LuxG from <i>Photobacterium leiognathi</i> TH1 and LuxABs from both <i>P. leiognathi</i> TH1 and <i>Vibrio campbellii</i> (<i>Pl</i>LuxAB and <i>Vc</i>LuxAB, respectively) was investigated using single-mixing and double-mixing stopped-flow spectrophotometry. The oxygenase component of <i>p</i>-hydroxyphenylacetate hydroxylase (C2) from <i>Acinetobacter baumannii</i>, which has no structural similarity to LuxAB, was used to measure the kinetics of release of FMNH^– from LuxG. With all FMNH^– acceptors used (C₂, <i>Pl</i>LuxAB, and <i>Vc</i>LuxAB), the kinetics of FMN reduction on LuxG were the same, showing that LuxG releases FMNH^– with a rate constant of 4.5–6 s^–1. Our data showed that the kinetics of binding of FMNH^–to <i>Pl</i>LuxAB and <i>Vc</i>LuxAB and the subsequent reactions with oxygen were the same with either free FMNH^– or FMNH^– generated <i>in situ</i> by LuxG. These results strongly suggest that no complexes between LuxG and the various species are necessary to transfer FMNH^– to the acceptors. The kinetics of the overall reactions and the individual rate constants correlate well with a free diffusion model for the transfer of FMNH^– from LuxG to either LuxAB

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

<div><p>3,4-dihydroxyphenylacetate (DHPA) dioxygenase (DHPAO) from <i>Pseudomonas aeruginosa</i> (PaDHPAO) was overexpressed in <i>Escherichia coli</i> 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 <i>K</i>_m for DHPA of 58 ± 8 μM and a <i>k</i>_cat 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 p<i>K</i>_a 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(<i>k’</i>_cat) <i>versu</i>s the reciprocal of the absolute temperature shows two correlations with a transition temperature at 35°C. Two activation energy values (<i>E</i>_a) 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.</p></div

Molecular mass of purified PaDHPAO.

<p>(A) SDS-PAGE (12%(w/v)) of the purified PaDHPAO. Lane 1 is a protein molecular weight standard marker (kDa) (Enzmart Biotech, Thailand) and lane 2 is an enzyme solution after purification by Phenyl-Sepharose chromatography. (B) A plot of relative volumes (V_e/V_o) <i>versus</i> the logarithms of the known molecular masses of protein standards (□): (1) ferritin (440 kDa), (2) aldolase (158 kDa), (3) BSA (65.4 kDa), (4) ovalbumin (48.9 kDa), (5) chymotrypsinogen (22.8 kDa), (6) ribonuclease (15.8 kDa), and PaDHPAO (●).</p