Probing the functionalities of alphaGlu328 and alphaAla74 of Vibrio harveyi luciferase by site-directed mutagenesis and chemical rescue.
This work aimed at identifying essential residues on the alpha subunit of Vibrio harveyi luciferase and elucidating their functional roles. Four conserved alpha-subunit residues at the proposed luciferase active site were initially mutated to Ala. Screening of the in vivo bioluminescence of cells expressing these mutated luciferases allowed the work to focus on alphaGlu328 for additional mutations to Phe, Leu, Gln, His, and Asp. V. harveyi luciferase is known to contain, at the same proposed active site, an unusual cis-peptide linkage between alphaAla74 and alphaAla75. To explore the structure-function relationship, luciferase variants alphaA74F and alphaA74G were constructed. The six alphaGlu328-mutated and the two alphaAla74-mutated luciferase variants were purified and characterized with respect to Vmax, Michaelis constants, light and dark decays, quantum yield, and, for alphaE328F and alphaA74F, yield of the 4a-hydroperoxyFMN intermediate and the ability to oxidize aldehyde substrate. Results indicated that the structural integrities of both alphaGlu328 and alphaAla74 were essential to luciferase bioluminescence activity. Moreover, the essentiality of alphaGlu328 was linked to the acidic nature of its side chain. The low activity of alphaE328A was sensitive to chemical rescue by sodium acetate, an effect that was not reproduced by phosphate. The efficiency of activity rescue by acetate progressively increased at lower pH in the range from 6.0 to 8.0, supporting the interpretation of alphaGlu328 as a catalytic general acid. The rescuing effect of acetate was on a reaction step after the formation of the 4a-hydroperoxyFMN intermediate. The exact catalytic function of alphaGlu328 is unclear, but possibilities are discussed.
Li
Chi-Hui
CH
Department of Biology and Biochemistry, University of Houston, Houston, Texas 77204-5001, USA.
Tu
Shiao-Chun
SC
D000409:Alanine / Q000737:chemistry*; D000447:Aldehydes / Q000378:metabolism; D002384:Catalysis; D018698:Glutamic Acid / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008156:Luciferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015335:Molecular Probes; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D014733:Vibrio / Q000201:enzymology*
D000447:Aldehydes; D015335:Molecular Probes; D018698:Glutamic Acid; D008156:Luciferases; D000409:Alanine