Structural and mechanistic analysis of trichodiene synthase using site-directed mutagenesis: probing the catalytic function of tyrosine-295 and the asparagine-225/serine-229/glutamate-233-Mg2+B motif.
Trichodiene synthase from Fusarium sporotrichioides contains two metal ion-binding motifs required for the cyclization of farnesyl diphosphate: the "aspartate-rich" motif D(100)DXX(D/E) that coordinates to Mg2+A and Mg2+C, and the "NSE/DTE" motif N(225)DXXSXXXE that chelates Mg2+B (boldface indicates metal ion ligands). Here, we report steady-state kinetic parameters, product array analyses, and X-ray crystal structures of trichodiene synthase mutants in which the fungal NSE motif is progressively converted into a plant-like DDXXTXXXE motif, resulting in a degradation in both steady-state kinetic parameters and product specificity. Each catalytically active mutant generates a different distribution of sesquiterpene products, and three newly detected sesquiterpenes are identified. In addition, the kinetic and structural properties of the Y295F mutant of trichodiene synthase were found to be similar to those of the wild-type enzyme, thereby ruling out a proposed role for Y295 in catalysis.
Vedula
L Sangeetha
LS
Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323, USA.
Jiang
Jiaoyang
J
Zakharian
Tatiana
T
Cane
David E
DE
Christianson
David W
DW
D020816:Amino Acid Motifs; D001216:Asparagine / Q000737:chemistry; D001671:Biochemistry / Q000379:methods*; D019755:Carbon-Carbon Lyases / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism; D005670:Fusarium / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry*; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D012694:Serine / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
D018698:Glutamic Acid; D014443:Tyrosine; D012694:Serine; D001216:Asparagine; D019755:Carbon-Carbon Lyases; C051130:trichodiene synthetase; D008274:Magnesium