Chiral deaza-coelenterazine analogs for probing a substrate-binding site in the Ca2+-binding photoprotein aequorin

Abstract

The Ca2+-binding photoprotein aequorin is a complex of apoAequorin (apoprotein) and (S)-2-peroxycoelenterazine. Aequorin can be regenerated by the incubation of apoAequorin with coelenterazine and molecular oxygen (O2). In this study, to investigate the molecular recognition of apoAequorin for coelenterazine using chemical probes, the chiral deaza-analogs of (S)- and (R)-deaza-CTZ (daCTZ) for coelenterazine and of (S)-2- and (R)-2-hydroxymethyl-deaza-CTZ (HM-daCTZ) for 2-peroxycoelenterazine were efficiently prepared by the improvement method. The chiral deaza-analogs of (S)-daCTZ and (S)-HM-daCTZ selectively inhibited the regeneration step to aequorin by binding the catalytic site of coelenterazine in the apoAequorin molecule. The crystal structures of the apoAequorin complexes with (S)-daCTZ and (S)-HM-daCTZ were determined, suggesting that the hydroxy moiety at the C6-hydroxyphenyl group and the carbonyl moiety of the imidazopyrazinone ring in coelenterazine are essential to bind the apoAequorin molecule through hydrogen bonding. Therefore, the chiral deaza-analogs of coelenterazine can be used as a probe to study the interaction between coelenterazine and the related proteins including photoprotein, luciferase, and coelenterazine-binding protein.

A catalytic mechanism for Renilla-type bioluminescence

“…A similar strategy using a different CTZ derivative was recently used to probe the substrate-binding site and mechanism of the Ca 2+ -regulated photoprotein aequorin. 55 We have also proposed a detailed reaction mechanism for Renilla -type bioluminescence that was inferred by considering multiple co-crystal structures of AncFT and RLuc8 luciferases and is supported by the results of mutagenesis, spectroscopic, and computational experiments. We show that CTZ adopts a Y-shaped conformation in the active sites of these enzymes, which is required for proper positioning of its imidazopyrazinone core in the enzymatic pocket.…”

Reverse mutants of the catalytic 19 kDa mutant protein (nanoKAZ/nanoLuc) from Oplophorus luciferase with coelenterazine as preferred substrate

“…Interestingly, even though CTZ is not a suitable substrate in nanoKAZ [ 5 , 9 ], CTZ was completely consumed and converted to CTMD and CTM with 63% and 37% in 2 h, respectively ( Table 6 ). As we recently reported, the products after incubation of CTZ (473 pmol) in 50 mM Tris-HCl (pH 7.6) at 25°C for 2 h in the absence of luciferase were CTZ (149 pmol), CTMD (68 pmol), CTM (65 pmol), dCTZ (90 pmol) and an unknown product ( Table 6 ) [ 19 ]. Because the formation of dCTZ was not detected in the reaction mixtures of CTZ with nanoKAZ and other luciferases, the CTM and CTMD formation from CTZ by nanoKAZ could be an enzymatic oxidation process.…”

“…6h- Coelenterazine ( 6h- CTZ), 6h-f- coelenterazine ( 6h-f -CTZ), and furimazine (FMZ) were synthesized as previously reported [ 5 ] ( Fig 1B ). The syntheses of the C2-modified CTZ analogs [ 20 ] ( Fig 1B ) and deaza-CTZ analogs [ 19 ] ( Fig 1C ) were described in our previous reports. Oligonucleotides used for site-directed mutagenesis and the synthetic gene of SNH-nanoKAZ (a nanoKAZ mutant with three amino acid substitutions; D19 S , D85 N , and C169 H ) which has the identical amino acid sequence to teLuc [ 15 ]), were obtained from Eurofins Genomics (Tokyo, Japan).…”

“…The reaction mixture containing each CTZ-utilizing luciferase (5 μg/5 μL) and CTZ (2 μg = 473 pmol/μL dissolved in ethanol) in 100 μL of 50 mM Tris-HCl (pH 7.6) was incubated at 25°C for 2 h and then was added to 100 μL of 6 M guanidine-HCl in H 2 O (pH 7.3) (Wako Pure Chemicals), and was extracted with 500 μL of diethyl ether using a vortex mixer for 15 s. After centrifugation at 10,000 rpm for 3 min at room temperature, 450 μL of ether layer was recovered and was dried down in vacuo . The resultant residues were dissolved in 100 μL of ethanol, showing purple-blue fluorescence under a 365 nm lamp, and 10 μL of the solution was subjected to HPLC analysis for CTZ, CTMD, CTM, CTO, and dCTZ [ 19 , 28 ]. An Agilent (CA, USA) 1200 series HPLC system was used under the following conditions: column, Wakosil 5C4 (ø4.6 mm × 250 mm); eluting solvent, CH 3 CN/H 2 O containing 0.1% trifluoroacetic acid; gradient elution, 40% CH 3 CN for 10 min, 40–50% CH 3 CN for 20 min, 50–80% CH 3 CN for 10 min, and 80% CH 3 CN for 10 min; flow rate, 0.5 mL/min; column temperature, 25°C; and detector, 225, 280, 330 and 450 nm using a diode array detector.…”

“…The luminescence properties of QL-nanoKAZ were characterized and compared with other CTZ-utilizing luciferases, including native OpLase [ 2 ], nanoKAZ [ 5 ], nanoKAZ mutant with three amino acids substitutions (SNH-nanoKAZ = teLuc) [ 15 ], GLase [ 16 ], RLase [ 17 ], and RLase mutant with red-shifted luminescence spectrum (RLase-547 = RLuc8.6–547) [ 18 ]. Furthermore, the crystal structure of QL-nanoKAZ was determined (PDB ID: 7VSX) and the substrate recognition of QL-nanoKAZ for CTZ was discussed based on the results of substrate specificity for CTZ analogs and luminescence inhibition with deaza-coelenterazine analogs [ 19 ].…”