HOMOLOGY MODELING OF NEUROSPORA-CRASSA GERANYLGERANYL PYROPHOSPHATE SYNTHASE - STRUCTURAL INTERPRETATION OF MUTANT PHENOTYPES

A model of the tertiary structure of the Neurospora crassa carotenogen ic prenyltransferase, geranylgeranyl pyrophosphate synthase (GGPPS), i s presented, based on structural homology with other prenyltransferase s and on the crystal structure of recombinant avian farnesyl pyrophosp hate synthase (FPPS). The conserved aspartate-rich moths DDxx(xx)D and associated basic residues, considered to be the active sites for bind ing and catalysis in all prenyltransferases, are highly conserved in t he N.crassa GGPPS protein, while other regions display a lower degree of sequence homology; thus the GGPPS model structure is predicted to b e highly reliable in the active site region. A number of carotene-defi cient mutants have been generated utilizing the repeat-induced point m utation (RIP) mechanism: mutant al-3(RIP1) carries a Ser-to-Asn mutati on in position 336 which falls within the predicted active site of the enzyme, Analysis of the model structure of this mutant indicates that Ser336 may be involved in substrate uptake, Two other mutants, al-3(R IP3) and al-3(RIP6), carry mutations in positions in the GGPPS protein , homologous to regions of the avian FPPS enzyme proposed to be involv ed in enzyme dimerization and substrate uptake, respectively, suggesti ng an explanation for the reduced carotene content of these mutants.