Tryptophan 250 on the alpha subunit plays an important role in flavin and aldehyde binding to bacterial luciferase. Effects of W-->Y mutations on catalytic function.
Bacterial luciferase is a heterodimer (alpha beta) that catalyzes the oxidation of FMNH2 and a fatty aldehyde, resulting in light emission. To explore the nature of the flavin binding site with respect to the role of tryptophan residues, the catalytic and binding properties of single-point mutants of Xenorhabdus luminescens luciferase with one of the eight tryptophans converted to a tyrosine residue were investigated by luminescence and fluorescence measurements. Conversion of tryptophans 194 and 250 on the alpha subunit to tyrosine had relatively large effects on the properties of luciferase with only minor changes in the properties on mutation of the other four tryptophans on alpha and the two on the beta subunit. Mutation of alpha W250 decreased the binding to FMNH2, FMN, aldehyde, and fatty acid, causing major changes in luminescence emission and decay. The results are consistent with alpha W250 interacting with flavin which in turn affects aldehyde binding. Mutation of alpha W194 did not affect the interaction with flavin or aldehyde but did change the relative rate of decay of light emission with aldehydes of different chain lengths as well as the activation energy for this process. Moreover, these results provide evidence for alpha W250, and to a lesser extent alpha W194, being in contact with the isoalloxazine ring of flavin, a proposal that has been recently made based on a model with flavin bound to the alpha subunit and anchored at a binding site for the phosphate moiety of FMN(H2) identified in the crystal structure of Vibrio harveyi luciferase [Fisher, A. J. Raushel, F. M., Baldwin, T. O., & Rayment, I. (1995) Biochemistry 34, 6581-6586].
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Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
Meighen
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D000447:Aldehydes / Q000378:metabolism*; D001665:Binding Sites; D004755:Enterobacteriaceae / Q000201:enzymology; D005227:Fatty Acids / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000378:metabolism*; D007700:Kinetics; D008156:Luciferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements; D016296:Mutagenesis; D010084:Oxidation-Reduction; D017354:Point Mutation; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism*; D014443:Tyrosine
D000447:Aldehydes; D005227:Fatty Acids; D014443:Tyrosine; D005486:Flavin Mononucleotide; D014364:Tryptophan; D008156:Luciferases