The Luciferin Binding Site Residues C/T311 (S314) Influence the Bioluminescence Color of Beetle Luciferases through Main-Chain Interaction with Oxyluciferin Phenolate

Author(s)

Viviani, V. R. ^{[1, 2]} ; Amaral, D. T. ^{[1, 2]} ; Neves, D. R. ^[1] ; Simoes, A. ^[1] ; Arnoldi, F. G. C. ^[3]

Affiliation

^[1] Fed Univ Sao Carlos UFSCAR, Dept Phys Chem & Math, Grad Program Biotechnol & Environm Monitoring, Sorocaba, SP - Brazil

^[2] Fed Univ Sao Carlos UFSCAR, Grad Program Evolut Genet & Mol Biol, Sao Carlos, SP - Brazil

^[3] Univ Sao Paulo, Ribeirao Preto Sch Med, BR-14049 Ribeirao Preto, SP - Brazil

• Document type: Journal article
• Source: BIOCHEMISTRY; v. 52, n. 1, p. 19-27, JAN 8 2013.
• Web of Science Citations: 14

Abstract

Beetle luciferases emit different bioluminescence colors from green to red; however, no clear relationship between the identity of the luciferin binding site residues and bioluminescence colors was found in different luciferases, and it is unclear whether critical interactions affecting emission spectra occur on the thiazolyl or on the benzothiazolyl sides of the luciferin binding site. Through homology modeling and site-directed mutagenesis using our multicolor set of beetle luciferases (Pyrearinus termitilluminans larval click beetle, Pte, lambda(max) = 534 nm; Phrixothrix hirtus railroad worm red emitting, PxRE, lambda(max) = 623 nm; and Macrolampis sp2 firefly, Mac, lambda(max) = 564 nm), we show that the residues C/T311 (S314) play an important role in bioluminescence color determination. Modeling studies indicate that the main-chain carbonyls of these residues are close to both oxyluciferin phenolate and AMP, whereas the side chains pack against second-shell residues. The C311(S314)A mutation considerably red shifts the spectra of the green-yellow-emitting luciferases (Pte lambda(max) = 534 to 590 nm; Mac lambda(max) = 564 to 583/613 nm) and affects the Km values for luciferin and ATP, but not the spectrum of the red-emitting luciferase. On the other hand, whereas the exchange between C/T311 (S314) caused smaller effects on the emission spectra of green-yellow-emitting luciferases, the C311T substitution (naturally found in green-emitting railroad worm luciferases) resulted in the largest reported blue shift in P. hirtus red-emitting luciferase (lambda(max) = 623 to 606 nm). Altogether, these results indicate that the stability of residues C/T311 (S314) and the size of the cavity around oxyluciferin phenolate affect bioluminescence colors and suggest, for the first time, the occurrence of a critical interaction between main-chain carbonyls of position 311 (314) residues and oxyluciferin phenolate. (AU)

• FAPESP's process: 11/23961-0 - pH-sensitive luciferases: structural origin of pH-sensitivity, and suitability of use in cell biosensors for pH and heavy metals
• Grantee: Vadim Viviani
• Support Opportunities: Regular Research Grants
• FAPESP's process: 12/04857-0 - Structural origin and evolution of luciferase/oxigenase activity in AMP-CoA-ligases and beetle luciferases
• Grantee: Vadim Viviani
• Support Opportunities: Regular Research Grants