Introduction
Luciferases are a class of enzymes that catalyze the oxidation of a long chain aliphatic aldehydes the emission of blue-green light. The luciferase found in 'Vibrio harveyi' is a heterodimer that is composed of a catalytic α subunit and a homologous but noncatalytic β subunit. This reaction results in the formation of a carboxylic acid, reduced flavinmononucleotide and the emission of photons in the form of blue-green light. The catalytic α subunit houses the active site and is connected to the β subunit via a single interatcion between the mobile loop and the α subunit at α Phe 272 and Tyr 151 of the β subunit.
Mechanism
Luciferase found in'V. Harveyi' binds noncovalently to a reduced flavin mononucleotide cofactor, an aliphatic aldehyde and oxygen to yield oxidized flavin mononucleotide, water, and carboxylic acid. The reaction occurs in two steps forming a hydroxyflavin intermediate and ultimately results in the oxidation of the aldehyde and emission of photons
FMNH2+O2+RCHO→FMN+RCOOH+H2O+hv(490nm)
The catalytic α subunit houses the FMN cofactor and is connected to the β subunit via a hairpin structure called the "." The organic substrate for bacterial luciferase in vivo is myristic aldehyde, although many aliphatic aldehydes of various lengths can induce bioluminescence in vitro[1]. Oxygen is needed for light generation, no bioluminescent activity occurs in anaerobic conditions[1].
Structural Motifs
Structure homology-There is a great deal of sequence homology and structural coservation between the α and β subunits. When superimposed over the barrels of the alpha and beta subunits with a deviation of 0.62Å for 42 equivalent α carbons. The region of the beta subunit that contains the 29 residue deletion with respect to the alpha subunit differs notably in arrangement. In the alpha subunit, the α7a helix is straight and extends toward the beta subunit. The region involved with dimerization, helices α and β are exceptionally similar in superposition.
Active Site and Alpha Subunit-the of bacterial luciferase is a large open cavity that is accessible to solvent via an opening located at the C-terminal ends of the ǰ strans of the TIM-barrel structure. During the first step of the oxidation reaction, FMNH2 binds to the flavin binding pocket and the enzyme undergoes a conformational change that blocks water in the surrounding environment from accessing both the excited peroxydihydroflavin intermediate. Next O2 and a long chain aldehyde bind to the FMNH2 luciferase complex and a two step oxidatino reaction occurs.
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The β subunit-The beta subunit is characterized as a necessary but non-catalytic subunit that stabilizes the catalytic ǯ subunit that is responsible for the oxidation reaction. The beta and alpha subunits are connected by a single interaction between the
Mobile Loop- Residues 272-288 on the α are known as the mobile loop. This portion of the alpha subunit contains a single residue that forms a salt bridge with the beta subunit and stabilizes the active site[2].
(β/α)8 TIM Barrel- The tertiary structure of the α and β subunits is very similar. While both the alpha and beta subunits are similar, the alpha subunit contains an extra 29 residues that the beta lacks. Both subunits fold into a single-domain eight-stranded β/α barrel motif. the two subunits assemble around a parallel four-helix bundle centered on a pseudo 2-fold axis that relates the alpha and beta subunits[3].
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