Mutation of a Protease-sensitive Region in Firefly Luciferase Alters Light Emission Properties

Abstract

Luciferase (EC 1.13.12.7) from the North American firefly, Photinus pyralis, is widely used as a reporter enzyme in cell biology. One of its distinctive properties is a pronounced susceptibility to proteolytic degradation that causes luciferase to have a very short intracellular half-life. To define the structural basis for this behavior and possibly facilitate the design of more stable forms of luciferase, limited proteolysis studies were undertaken using trypsin and chymotrypsin to identify regions of the protein whose accessible and flexible character rendered them especially sensitive to cleavage. Regions of amino acid sequence 206 -220 and 329 -341 were found to be sensitive, and because the region around 206 -220 had high homology with other luciferases, CoA ligases, and peptidyl synthetases, this region was selected for mutagenesis experiments intended to determine which of its amino acids were essential for activity. Surprisingly, many highly conserved residues including Ser 198 , Ser 201 , Thr 202 , and Gly 203 could be mutated with little effect on the luminescent activity of P. pyralis luciferase. One mutation, however, S198T, caused several alterations in enzymatic properties including shifting the pH optimum from 8.1 to 8.7, lowering the K m for Mg-ATP by a factor of 4 and increasing the half-time for light emission decay by a factor of up to 150. While the S198T luciferase was less active than wild type, activity could be restored by the introduction of the additional L194F and N197Y mutations. In addition to indicating the involvement of this region in ATP binding, these results provide a new form of the enzyme that affords a more versatile reporter system. Luciferase (EC 1.13.12.7) from the North American firefly, Photinus pyralis, is widely used as a reporter enzyme in studies of gene expression (1). Firefly luciferase first catalyzes the formation of a bound luciferin-AMP, which then reacts with O 2 to generate oxyluciferin-AMP in an excited state. Breakdown of this intermediate to oxyluciferin, CO 2 , and AMP is accompanied by the emission of a photon. The high quantum yield for this process, nearly a single photon emitted per reacted luciferin molecule (2), reflects not only an efficient catalytic machinery but also a highly favorable environment for radiative decay of an excited state.The kinetics of light emission can be modulated by coenzyme A, but this is not a required substrate (3, 4). Luciferase can also function as a ligase (5) as the AMP group of the enzyme⅐luciferyl-AMP complex can be transferred to ATP to produce diadenosine tetraphosphate.Despite many studies, relatively little was known about the structure of luciferase or the nature of its active site until recently. Cloning and sequencing of luciferase from the firefly (6) and its homologues from several beetles (reviewed in Ref . 7) have shown that these enzymes are related to certain acyltransferases (8, 9). Alignment of these related sequences reveals conserved residues that may be important for enzymatic activity. For exam...

Molecular basis for the high-affinity binding and stabilization of firefly luciferase by PTC124

“…Thermal denaturation assays of FLuc in the presence of PTC124-AMP show a large ΔT m of nearly 30°C, significantly greater than the sum of the ΔT m values for PTC124 and ATP at the same concentration, suggesting that the formation of the adduct is essential for the observed increase in protein stability. Analogs containing nonreactive acid isosteres at the meta position (3)(4)(5), and carboxylate-containing regioisomers at the ortho (10) or para (7) positions, also have significantly reduced thermal shifts, further indicating that FLuc protein stability is dependent on PTC124-AMP formation.…”

“…Although ideal for following dynamic responses in cell-based assays, reporter instability can be offset by ligand-induced stabilization. Stabilization of FLuc by compounds acting as inhibitors can lead to a relative increase in enzyme levels (compared to untreated basal levels) and correspondingly increased FLuc activity-a counterintuitive result for compounds characterized as FLuc inhibitors (5)(6)(7)(8).…”

“…However, as an enzyme that catalyzes the bimolecular reaction between two small molecule substrates, D-luciferin and ATP, it is prone to inhibition by a variety of low-molecular-weight heterocyclic compounds typically found in screening collections (3,4). Expressed intracellularly as a reporter, the FLuc enzyme has a short protein half-life (t 1∕2 ∼ 3-4 h) relative to other reporters (5,6). Although ideal for following dynamic responses in cell-based assays, reporter instability can be offset by ligand-induced stabilization.…”

Thermostabilization of Firefly Luciferases Using Genetic Engineering

“…This effect is especially pronounced at high temperatures when partial enzyme unfolding leads to the higher accessibility of proteolytic sites (Frydman, 1999). Two protease sensitive regions (206-220 and 329-341 residues) were identified in P. pyralis luciferase amino acid sequence (Thompson et al, 1997). Half-life of luciferase activity in vivo in mammalian cells is only 3-4 hours at 37°C (Leclerc et al, 2000;Thompson et al, 1991).…”

Identification of residues essential for a two-step reaction by malonyl-CoA synthetase from Rhizobium trifolii

“…A majority of the conserved residues are clustered on the surfaces of the two domains opposite each other, suggesting this as a probable location of the active site. It was proposed that a peptide (244HHGF247) obtained through photo-inactivation by 2-(4-benzoylphenyl)-thiazole-4-carboxylic acid (BPTC), a luciferin analogue, may be located at or near the luciferin-binding site [8]. It was subsequently reported that luciferase His-245 mutants showed extremely low specific activity, suggesting that the histidine residue plays a critical role in the active site [9].…”