Much like the similar bacterial haloalkane dehalogenase [32
luciferase has a characteristic α
-hydrolase fold sequence at its core [33
] and shares the conserved catalytic triad of residues employed by the dehalogenases [22
]. The level of primary sequence similarity is somewhat surprising given that the dehalogenases are hydrolases while the luciferase is an oxidase, and hypotheses on how this situation may have came to be from an evolutionary standpoint have previously been discussed [22
]. Now with the structure of the luciferase, the high level of tertiary structure similarity to the haloalkane dehalogenases can be noted as well (Cα
root mean square deviations ~1.5 Å for LinB structures). A topological map of the RLuc8 α
-hydrolase fold is shown in , along with the locations of the presumptive catalytic triad residues D120/E144/H285A within this diagram. α
-hydrolases have their nucleophile (D120 in RLuc) immediately after the fifth β
5) in what is termed the “nucleophile elbow”. The sequence pattern for this elbow is generally G-X-Nuc-X-G [34
] in the α
-hydrolases, and corresponds to GHDWG (residues 118–122) in Renilla
Figure 3 3a The topology of RLuc8’s α/β-hydrolase fold domain. α-helices are shown in blue, and β-sheets are shown in red. Numbering/lettering of the sheets/helices is done with respect to the standard for α/β (more ...)
A feature observable in the RLuc8 structures () that was not apparent in previous homology models is the wrapping of the N-terminus around the protein toward the front of the presumptive enzymatic pocket. There was also occasional variability in the placement of the initial ~10–15 residues (e. g. monomer 1 in ), indicating that this region of the protein may contain a high degree of conformational flexibility. Variants of RLuc8 with the N-terminal clipped up to position I15 are relatively well tolerated and retain >25% of activity (data not shown, see [24
]), demonstrating that this N-terminal region is not required for enzymatic activity of the protein. It has been a general observation in our laboratory that fusion proteins created by attachment to the N-terminus of RLuc have a greater propensity towards low luciferase activity than fusions made by attachment to the C-terminus of RLuc. Based on the structural data and the non-essentialness of the N-terminal region from an enzymatic standpoint, it can be hypothesized that this drop in activity for N-terminal fusions is due to steric hindrance of the RLuc active site.
In the RLuc8:diammonium structure two imidazole molecules, apparently from the mother liquor, were located in the presumptive catalytic pocket of the molecule. Previous reports [35
] have reported enhanced enzymatic activity of RLuc in the presence of imidazole, with a maximal activity enhancement of 2-fold at ~4 mM. While the reason for this potentiation remains unclear, a plausible explanation is that imidazole maintains the enzymatic pocket in a conformation appropriate for binding coelenterazine. Interestingly, the mother liquor for the RLuc8:KSCN condition did not include imidazole, although density corresponding to a single molecule of imidazole was present in the diffraction data from this condition. This indicates that the imidazole molecule is retained within the protein during the nickel affinity purification, and is bound tightly enough to remain attached through two additional steps of chromatography.
The main conformation changes in the coelenteramide-bound structure from the RLuc8:PEG/isopropanol condition compared to the previous diammonium phosphate condition, were a slight outward shift of the residues F261/F262/S263 and a larger outward movement of residues from W153 to A163. Residues 153–163 are within the cap domain () of the enzyme, a domain that has been suggested to be flexible for the purposes of substrate binding in the haloalkane dehalogenases [36
]. It can be expected that portions of the cap domain in Renilla
luciferase, specifically residues 153–163, are similarly flexible for this same purpose. The finding of flexibility is further supported by the high B-factors found for these residues, and the outward movement of this portion of the enzyme may indicate conformational changes in response to binding of the coelenteramide.
Several lines of evidence indicate that the observed location for coelenteramide in the RLuc8:PEG/isopropanol structure is not the location of the substrate during the enzymatic reaction nor the location of the product during emission of the bioluminescence photon. First, if the coelenteramide location shown in was the catalytic location, it would seem to indicate that two monomers of RLuc are involved in the enzymatic reaction. The reaction rate of RLuc, however, is first order with respect to enzyme concentration (Supplemental Figure 2
) indicating that only one protein monomer is involved in the reaction. Second, a variant with truncation of the last 5 amino acids of the protein (including the residue N309) retains 40% of the enzymatic activity (data not shown, see [24
]). Third, the K25A/E277A mutations resulted in only a 40% drop in activity (Supplemental Table 1
). One might expect a much larger drop in activity if E277 was directly involved in the enzymatic activity. Finally, many of the residues in the putative active pocket identified as being important for activity (e. g. N53, D120, I223 [22
]) would be rather distant (6–8 Å ) from the substrate/product if the observed coelenteramide location was correct.
It has been previously noted that the fluorescent emission spectrum of RLuc mixed with coelenteramide does not reconstitute the recorded bioluminescence emission spectrum [5
]. Coelenteramide, however, is known to strongly inhibit the enzymatic reaction with a Ki
~20 nM [37
], so it must be able to bind to RLuc tightly. The explanation for this phenomenon has been that the chemical environment coelenteramide experiences changes immediately following emission of the bioluminescence photon [35
]. This in turn leads us to the hypothesis that the coelenteramide location changes immediately after the enzymatic reaction and emission of the bioluminescence photon, with the coelenteramide sliding partially out of the active pocket due to a conformational change in the luciferase’s cap domain. In this hypothesis, the location of coelenteramide in the crystal structure presented here represents this “secondary” binding position and not the position of product/substrate during the enzymatic reaction.
Previously reported random and semi-rational mutation experiments on RLuc have been able to alter the enzyme’s substrate specificity [22
] as well as shift its bioluminescence emission spectra [23
]. While the coelenteramide bound structure of RLuc8 cannot fully explain these alterations, the structure does highlight many of the residue locations previously found to be most important in altering its emission spectrum (e. g. D162, F181, F261, F262) and should serve as a initial starting point for further rational alteration of the enzyme.
These previous reports have also yielded extensive data as to the enzymatic effects of mutagenesis on presumptive active site residues. This data, displayed diagrammatically in , shows that the residue locations where mutagenesis most effects enzymatic activity (N53, D120, W121, E144, P220, H285) are clustered toward the back of the active pocket. These 6 critical residues include the aforementioned catalytic triad, and are likely involved in coordinating the attack of the coelenterazine molecule by molecular oxygen during catalysis or alternatively may form an adduct with the coelenterazine. Around this core of critical resides, is a surrounding ring of less critical hydrophobic and aromatic residues (predominantly isoleucines, valines, phenylalanines, and trytophans). This ring is presumptively involved in assuring specificity when binding the hydrophobic coelenterazine molecule and orientating it with respect to the catalytic residues.
Further studies will be needed to elucidate the exact enzymatic mechanism of Renilla
luciferase and why its catalyzed reaction differs from that of the haloalkane dehalogenases despite their structural similarity. A number of mechanism-based coelenterazine analog inhibitors have been previously synthesized [38
] and may prove useful for both future crystallography work and for the study of the luciferase’s enzymatic kinetics. Additionally, work is ongoing using anoxic conditions in an attempt to crystallize the luciferase with its substrate.