Conversion of Product Specificity of Archaebacterial Geranylgeranyl-diphosphate Synthase : IDENTIFICATION OF ESSENTIAL AMINO ACID RESIDUES FOR CHAIN LENGTH DETERMINATION OF PRENYLTRANSFERASE REACTION

The most important function of carotenoid pigments, especially beta-carotene in higher plants, is to protect organisms against photooxidative damage (G. Britton, in T. W. Goodwin, ed., Plant Pigments--1988, 1988; N. I. Krinsky, in O. Isler, H. Gutmann, and U. Solms, ed., Carotenoids--1971, 1971). beta-Carotene also functions as a precursor of vitamin A in mammals (G. A. J. Pitt, in I. Osler, H. Gutmann, and U. Solms, ed., Carotenoids--1971, 1971). The enzymes and genes which mediate the biosynthesis of cyclic carotenoids such as beta-carotene are virtually unknown. We have elucidated for the first time the pathway for biosynthesis of these carotenoids at the level of enzyme-catalyzed reactions, using bacterial carotenoid biosynthesis genes. These genes were cloned from a phytopathogenic bacterium, Erwinia uredovora 20D3 (ATCC 19321), in Escherichia coli and located on a 6,918-bp fragment whose nucleotide sequence was determined. Six open reading frames were found and designated the crtE, crtX, crtY, crtI, crtB, and crtZ genes in reference to the carotenoid biosynthesis genes of a photosynthetic bacterium, Rhodobacter capsulatus; only crtZ had the opposite orientation from the others. The carotenoid biosynthetic pathway in Erwinia uredovora was clarified by analyzing carotenoids accumulated in E. coli transformants in which some of these six genes were expressed, as follows: geranylgeranyl PPiCrtB----prephytoene PPiCrtE----phytoeneCrtI---- lycopeneCrtY----beta-caroteneCrtZ----zeaxanthinCrtX--- -zeaxanthin-beta- diglucoside. The carotenoids in this pathway appear to be close to those in higher plants rather than to those in bacteria. Also significant is that only one gene product (CrtI) for the conversion of phytoene to lycopene is required, a conversion in which four sequential desaturations should occur via the intermediates phytofluene, zeta-carotene, and neurosporene.

Isoprenyl diphosphate synthases are ubiquitous enzymes that catalyze the basic chain-elongation reaction in the isoprene biosynthetic pathway. Pairwise sequence comparisons were made for 6 farnesyl diphosphate synthases, 6 geranylgeranyl diphosphate synthases, and a hexaprenyl diphosphate synthase. Five regions with highly conserved residues, two of which contain aspartate-rich DDXX(XX)D motifs found in many prenyltransferases, were identified. A consensus secondary structure for the group, consisting mostly of alpha-helices, was predicted for the multiply aligned sequences from amino acid compositions, computer assignments of local structure, and hydropathy indices. Progressive sequence alignments suggest that the 13 isoprenyl diphosphate synthases evolved from a common ancestor into 3 distinct clusters. The most distant separation is between yeast hexaprenyl diphosphate synthetase and the other enzymes. Except for the chromoplastic geranylgeranyl diphosphate synthase from Capsicum annuum, the remaining farnesyl and geranylgeranyl diphosphate synthases segregate into prokaryotic/archaebacterial and eukaryotic families.

The molecular cloning and the determination of the nucleotide sequence of the ispA gene responsible for farnesyl diphosphate (FPP) synthase [EC 2.5.1.1] activity in Escherichia coli are described. E. coli ispA strains have temperature-sensitive FPP synthase, and the defective gene is located at about min 10 on the chromosome. The wild-type ispA gene was subcloned from a lambda phage clone containing the chromosomal fragment around min 10, picked up from the aligned genomic library of Kohara et al. [Kohara, Y., Akiyama, K., & Isono, K. (1987) Cell 50, 495-508]. The cloned gene was identified as the ispA gene by the recovery and amplification of FPP synthase activity in an ispA strain. A 1,452-nucleotide sequence of the cloned fragment was determined. This sequence specifies two open reading frames, ORF-1 and ORF-2, encoding proteins with the expected molecular weights of 8,951 and 32,158, respectively. A part of the deduced amino acid sequence of ORF-2 showed similarity to the sequences of eucaryotic FPP synthases and of crtE product of a photosynthetic bacterium. The plasmid carrying ORF-2 downstream of the lac promoter complemented the defect of FPP synthase activity of the ispA mutant, showing that the product encoded by ORF-2 is the ispA product. The maxicell analysis indicated that a protein of molecular weight 36,000, approximately consistent with the molecular weight of the deduced ORF-2-encoded protein, is the gene product.