Pigment stoichiometries in the D,/D&ytochrome 6-559 reaction center complex from spinach have been studied by spectrophotometry and normalphase silica HPLC of the solvent extracts. Based on the well-accepted molar ratio Pheo a/P680=2, the results are summarized by Chl a/P680 = 6.01 k 0.37 (n = 24) and b-carotene/P680 = 1.84 f 0.11 (n = 4). These stoichiometries are significantly different from those of bacterial reaction center complexes.

Prenyltransferases catalyze the consecutive condensations of isopentenyl diphosphate to produce linear polyprenyl diphosphates. Each enzyme forms the final product with a specific chain length. The product specificity of an enzyme is thought to be determined by the structure around the unknown path through which the product elongates in the enzyme. To explore the path, we introduced a few mutations at the 5th, the 8th, and/or the 11th positions before the first aspartate-rich motif of geranylgeranyl-diphosphate synthase or farnesyldiphosphate synthase. The side chains of these amino acids are situated on the same side of an ␣-helix. In geranylgeranyl-diphosphate synthase, a single mutated enzyme (F77S) mainly produces a C 25 product (Ohnuma, S.-I., Hirooka, K., Hemmi, H., Ishida, C., Ohto, C., and Nishino, T. (1996) J. Biol. Chem. 271, 18831-18837). A double mutated enzyme (L74G and F77G) mainly produces a C 35 compound with significant amounts of C 30 and C 40 . A triple mutated enzyme (I71G, L74G, and F77G) mainly produces a C 40 compound with C 35 and C 45 . Mutated farnesyl-diphosphate synthases also show similar patterns. These findings indicate that the elongating product passages on a surface of the side chains of the mutated amino acids, the original bulky amino acids had blocked the elongation, and the path is conserved in prenyltransferases. Moreover, the fact that some double and triple mutated enzymes can also form small amounts of products longer than C 50 indicates that the paths in these mutated enzymes can partially access the outer surface of the enzymes.Prenyltransferases, also referred to as polyprenyl-diphosphate synthases, are indispensable for biosyntheses of more than 20,000 naturally occurring isoprenoids and constitute a broad family of enzymes that catalyze the sequential condensations of isopentenyl diphosphate (IPP, C 5 ) 1 with allylic prenyl diphosphates (1). These enzymes are classified into two groups according to the stereochemistry of the E or Z double bond that is formed by the condensation. Although organisms use Z-polyprenyl-diphosphate synthases only for the synthesis of dolichols for N-linked glycoprotein biosynthesis, Z-polyprenols for peptidoglycan biosynthesis in bacteria, and natural rubber, E-polyprenyl-diphosphate synthases are used for the synthesis of a vast variety of important natural isoprenoids (Fig. 1). A number of enzymes that yield (all-E)-prenyl diphosphate have been isolated from various organisms. Geranyldiphosphate (GPP, C 10 ) synthase, found in plants (2), catalyzes the single condensation of IPP with dimethylallyl diphosphate (DMAPP, C 5 ) to give GPP. The GPP is the precursor of all monoterpenes. Farnesyl-diphosphate (FPP, C 15 ) synthase, which is one of the key enzymes of the biosynthesis of steroids, cholesterol, farnesylated proteins, sesquiterpenes, and so on, catalyzes the consecutive condensation of two molecules of IPP with DMAPP to give FPP as the ultimate product. Geranylgeranyl-diphosphate (GGPP, C 20 ) synthases are thought to be classifie...

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Prenyltransferases (prenyl diphosphate synthases), which are a broad group of enzymes that catalyze the consecutive condensation of homoallylic diphosphate of isopentenyl diphosphates (IPP, C5) with allylic diphosphates to synthesize prenyl diphosphates of various chain lengths, have highly conserved regions in their amino acid sequences. Based on the above information, three prenyltransferase homologue genes were cloned from a thermophilic cyanobacterium, Synechococcus elongatus. Through analyses of the reaction products of the enzymes encoded by these genes, it was revealed that one encodes a thermolabile geranylgeranyl (C20) diphosphate synthase, another encodes a farnesyl (C15) diphosphate synthase whose optimal reaction temperature is 60 degrees C, and the third one encodes a prenyltransferase whose optimal reaction temperature is 75 degrees C. The last enzyme could catalyze the synthesis of five prenyl diphosphates of farnesyl, geranylgeranyl, geranylfarnesyl (C25), hexaprenyl (C30), and heptaprenyl (C35) diphosphates from dimethylallyl (C5) diphosphate, geranyl (C10) diphosphate, or farnesyl diphosphate as the allylic substrates. The product specificity of this novel kind of enzyme varied according to the ratio of the allylic and homoallylic substrates. The situations of these three S. elongatus enzymes in a phylogenetic tree of prenyltransferases are discussed in comparison with a mesophilic cyanobacterium of Synechocystis PCC6803, whose complete genome has been reported by Kaneko et al. (1996).