The Neurospora crassa Carotenoid Biosynthetic Gene (Albino 3) Reveals Highly Conserved Regions among Prenyltransferases”

In the filamentous fungus Neurospora crassa the biosynthesis of carotenoids is regulated by blue light. Here we report the characterization of the albino-3 (al-3) gene of N. crassa, which encodes the carotenoid biosynthetic enzyme geranylgeranyl-pyrophosphate synthetase. This is the first geranylgeranyl-pyrophos-phate synthetase gene isolated. Nucleotide sequence comparison of al-3 genomic and cDNA clones revealed that the al-3 gene is not interrupted by introns. Transcription of the al-3 gene has been examined in dark-grown and light-induced mycelia. The analysis revealed that the al-3 gene is not expressed in the dark and that its transcription is induced by blue light (Nel-son, M. A., Morelli, G., Carattoli, A., Romano, N., and Macino, G. (1989) Mol.

Carotenoids are synthetized by bacteria, plants, fungi, and algae (2). While their primary functions are in photoprotection and as accessory pigments in photosynthesis, carotenoids also serve as precursors for vitamin A biosynthesis in animals and for abscissic acid biosynthesis in plants. In Neurospora crassa the biosynthesis of carotenoids is regulated by blue light in the mycelium but is constitutive in the asexual spores (3)(4)(5). The photoinduction of carotenogenesis in the mycelia requires the de nouo synthesis of at least three enzymes which have been shown to be the products of the albino (al) genes (5). Three a1 mutants have been characterized in N. crassa, each of which is defective in one step of carotenogenesis.
* This work was supported by grants from the Minister0 Agricoltura e Foreste, Piano Nazionale Tecnologie Applicate alle Piante, and Instituto Pasteur-Fondazione Cenci Bolognetti. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence($ reported in thispaper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) x53979.
Albino 3 (al-3) mutants are defective in GGPP' synthetase (5), while albino 2 and albino 1 mutants are defective in phytoene synthetase (6) and phytoene dehydrogenase (7), respectively. In previous work we isolated the gene encoding GGPP synthetase from N. crmsa by complementation of the al-3 mutant; expression studies showed that the transcription of the al-3 gene is controlled by light regulation (1).
The pathway of carotenoid biosynthesis shares some steps with the biosynthetic pathways of other isoprenoid compounds. GGPP synthetase and several other enzymes of these complex pathways are members of the prenyltransferase family. Prenyltransferases catalyze the transfer of an isoprenoid diphosphate to another isoprenoid diphosphate or to a nonisoprenoid compound through a 1'-4 condensation reaction to produce various prenyl compounds that are precursors of such diverse products as steroids, carotenoids, chlorophylls, heme a, prenylated proteins and tRNAs, glycosyl carrier lipids, plant hormones, and the side chains of quinones (8)(9)(10). Prenyltransferases produce a wide range of products, from the simple dimer geranyl pyrophosphate to the complex structure of rubber which is thousands of monomers long. GGPP synthetase catalyzes the trans addition of three molecules of IPP onto DMAPP to form geranylgeranyl pyrophosphate.
Here we present the sequence of the al-3 gene and its deduced amino acid sequence. The al-3 protein sequence is compared with those of other known prenyltransferases.

DISCUSSION
We have determined the nucleotide sequence of the al-3 gene, which encodes the carotenoid biosynthetic enzyme GGPP synthetase. It is known that blue light induces the biosynthesis of carotenoids in N. crmsa mycelia and that the activity of GGPP synthetase increases after light treatment (8). The al-3 gene encodes an mRNA of 1683 nucleotides, which is colinear with the al-3 gene, as revealed by cDNA sequence and S1 nuclease mapping analysis. We analyzed the expression of the al-3 gene and found that its mRNA is not present in dark-grown mycelia but is induced by blue light after a short pulse of illumination.
Portions of this paper (including "Materials and Methods," "Results," Table 1 The polypeptide encoded by the al-3 gene has a molecular mass of 47,876 daltons, is weakly basic and hydrophilic, and does not possess any hydrophobic membrane-spanning regions. This is in agreement with the finding that the GGPPS activity, isolated from various sources, is present in the soluble fraction of cellular extracts (8,36,37). GGPP synthetase is a prenyltransferase that catalyzes the 1'-4 condensation of dimethylallyl pyrophosphate with three isopentenyl pyrophosphates. We therefore compared the al-3 polypeptide with other known prenyltransferases. Comparison with the FPP synthetase from human (29), rat (30), and Saccharomyces cereuisiae (28) showed significant homologies in three different regions of these proteins. The relative positions of the homologous regions were the same in all the proteins analyzed. Furthermore, the three domains were localized in the proteins at very similar distances. These facts suggest that the homologous regions may be involved in the formation of the catalytic site of the prenyltransferases. Comparison of domains I and I11 showed the presence of the motif DDXXD.
These aspartate residues could be responsible for the binding of the cations Mg2+ or Mn2+, shown to be important for the catalytic activities of the prenyltransferases (38, 39). The analysis of the conserved amino acids found in the three domains suggested that in all three domains not only the aspartate residues but also the positively charged amino acids may be important for enzyme activity. One of the major biological functions of arginine residues is to interact with phosphorylated metabolites. Lysine residues may also serve this function and indeed are known to be important in a number of enzymes acting on phosphorylated substrates (40, 41). Furthermore, it has been demonstrated that the argininespecific reagent hydroxyphenylglyoxal is a powerful inhibitor of prenyltransferases (38, 42). Domain 11 is homologous to the region proposed to be the active site of avian FPP synthetase. Brems et al. (32) identified this region by a site-directed photoaffinity label on the purified enzyme and proposed that the arginine residue (whose position is indicated in Fig. 5 with an asterisk), conserved also in human and rat FPP synthetase (not shown) and substituted by lysine in all the other known prenyl transferases, could be responsible for the binding of the pyrophosphate group. These authors also showed evidence of the involvement of 2 arginine residues in the function of prenyltransferases.
In view of the model that we propose for the function of these conserved domains, it is interesting that the yeast prenyltransferase (involved in tRNA modification) has some homology, involving arginyl residues, with the first domain and a stronger homology with the third domain, in which all 3 aspartate residues are conserved.
We have found that the crtE gene product of Rhodobacter capsulatm also has an impressive homology with all three conserved domains. All the prenyltransferases considered here perform a 1 ' 4 condensation reaction between an allylic and an homoallylic substrate. CrtE has been indicated to be a phytoene synthetase (31) and therefore it does not belong to the prenyltransferase family. Due to these homologies, we propose that the crtE enzyme may have a 1'-4 condensation activity.
These similarities and homologies among prenyltransferases suggest that the genes may have a phylogenetic relation. It is conceivable that other prenyltransferases could share the same regions of homology with those considered here. The prediction that the conserved amino acids, in the three homologous regions, play an essential role in the enzyme functions, will be tested by site-directed mutagenesis experiments on the al-3 gene, making use of the albine 3 mutant as the recipient strain for the transformation of mutated sequences.  with probes a, b and c (Fig. 2). Results are sham. respectively. in panels A.