Isoprenoid phenol and quinone precursors of ubiguinones and dihydroubiguinones (ubiguinones (H 2 )) in fungi.

1. Ten moulds and two yeasts were analysed for the presence of 2-polyprenylphenols, 2-polyprenyl(H(2))phenols, 6-methoxy-2-polyprenylphenols, 6-methoxy-2-polyprenyl(H(2))phenols, 6-methoxy-2-polyprenyl-1,4-benzoquinones, 6-methoxy-2-polyprenyl(H(2))-1,4-benzoquinones, 5-demethoxyubiquinones, 5-demethoxyubiquinones(H(2)), ubiquinones and ubiquinones(H(2)). 2. The organisms were found to be of three types: (a) those that contained only ubiquinones (Aspergillus fumigatus and Penicillium brevi-compactum) or ubiquinones(H(2)) (Alternaria solani, Claviceps purpurae and Penicillium stipitatum); (b) those that contained 5-demethoxyubiquinones and ubiquinones (Agaricus campestris, Aspergillus niger, Phycomyces blakesleeanus, Rhodotorula glutinis and Saccharomyces cerevisiae) or 5-demethoxyubiquinones(H(2)) and ubiquinones(H(2)) (Aspergillus quadrilineatus and Neurospora crassa); (c) one that contained 2-decaprenyl(H(2))phenol, 6-methoxy-2-decaprenyl(H(2))phenol, 6-methoxy-2-decaprenyl(X-H(2))-1,4-benzoquinone, 5-demethoxyubiquinone-10(X-H(2)) and ubiquinones(H(2)) (Aspergillus flavus). 3. Studies were made on the biosynthesis of ubiquinones and ubiquinones(H(2)) by Asp. flavus, Phyc. blakesleeanus and S. cerevisiae. These provided evidence that in Phyc. blakesleeanus 5-demethoxyubiquinone-9 is a precursor of ubiquinone-9 and that in S. cerevisiae 5-demethoxyubiquinone-6 is a precursor of ubiquinone-6. In addition they yielded results that may be interpreted as providing evidence that in Asp. flavus 6-methoxy-2-decaprenyl(X-H(2))-1,4-benzoquinone and 5-demethoxyubiquinone-10(X-H(2)) are precursors of ubiquinone-10(X-H(2)).

Biosynthesis of phytoquinones. Homogentisic acid: a precursor of plastoquinones, tocopherols and alpha-tocopherolquinone in higher plants, green algae and blue-green algae.

1. By means of (14)C tracer experiments and isotope competition experiments the roles of d-tyrosine, p-hydroxyphenylpyruvic acid, p-hydroxyphenylacetic acid, phenylacetic acid, homogentisic acid and homoarbutin (2-methylquinol 4-beta-d-glucoside) in the biosynthesis of plastoquinones, tocopherols and alpha-tocopherolquinone by maize shoots was investigated. It was established that d-tyrosine, p-hydroxyphenylpyruvic acid and homogentisic acid can all be utilized for this purpose, whereas p-hydroxyphenylacetic acid, phenylacetic acid and homoarbutin cannot. Studies on the mode of incorporation of d-tyrosine, p-hydroxyphenylpyruvic acid and homogentisic acid showed that their nuclear carbon atoms and the side-chain carbon atom adjacent to the nucleus give rise (as a C(6)-C(1) unit) to the p-benzoquinone rings and nuclear methyl groups (one in each case) of plastoquinone-9 and alpha-tocopherolquinone and the aromatic nuclei and nuclear methyl groups (one in each case) of gamma-tocopherol and alpha-tocopherol. 2. By using [(14)C]-homogentisic acid it has been shown that homogentisic acid is also a precursor of plastoquinone, tocopherols and alpha-tocopherolquinone in the higher plants Lactuca sativa and Rumex sanguineus, the green algae Chlorella pyrenoidosa and Euglena gracilis and the blue-green alga Anacystis nidulans.

Biosynthesis of ubiquinone in non-photosynthetic gram-negative bacteria.

1. The polyprenylphenol and quinone complements of the non-photosynthetic Gram-negative bacteria, Pseudomonas ovalis Chester, Proteus mirabilis and ;Vibrio O1' (Moraxella sp.), were investigated. 2. Ps. ovalis Chester and Prot. mirabilis were shown to contain 2-polyprenylphenols, 6-methoxy-2-polyprenylphenols, 6-methoxy-2-polyprenyl-1,4-benzoquinones, 5-demethoxyubiquinones, ubiquinones, an unidentified 1,4-benzoquinone [2-polyprenyl-1,4-benzoquinone (?)] and ;epoxyubiquinones'. ;Vibrio O1' was shown to contain only 5-demethoxyubiquinones, ubiquinones and ;epoxyubiquinones'. 3. It was established that in Ps. ovalis Chester 2-polyprenylphenols, 6-methoxy-2-polyprenylphenols, 6-methoxy-2-polyprenyl-1,4-benzoquinones, 5-demethoxyubiquinones and 2-polyprenyl-1,4-benzoquinones (?) are precursors of ubiquinones. 4. Intracellular distribution studies showed that in Ps. ovalis Chester ubiquinone and its prenylated precursors are localized entirely on the protoplast membrane. 5. Investigations into the oxygen requirements for ubiquinone biosynthesis by Ps. ovalis Chester showed that the organism could not convert p-hydroxybenzoic acid into ubiquinone in the absence of oxygen, although it could convert a limited amount into 2-polyprenylphenols. 6. Attempts were made to prepare cell-free preparations capable of synthesizing ubiquinone. Purified protoplast membranes of Ps. ovalis Chester were found to be incapable of carrying out this synthesis, even when supplemented with cytoplasm. With crushed-cell preparations of Ps. ovalis Chester, organism PC4 (Achromobacter sp.) and Escherichia coli, synthesis was observed, although this was attributable in part to a small number of intact cells present in the preparations.

The nature, intergeneric distribution and biosynthesis of isoprenoid quinones and phenols in gram-negative bacteria.

1. Twenty-two aerobically grown Gram-negative bacteria were analysed for demethylmenaquinones, menaquinones, 2-polyprenylphenols, 6-methoxy-2-polyprenylphenols and ubiquinones. 2. All the eight enterobacteria and both the two facultative organisms (Aeromonas punctata and Aeromonas hydrophila) examined contain all the compounds listed above. The principal homologues are octaprenyl; in addition lower (down to tri- or tetra-prenyl for the 2-polyprenylphenols) and sometimes higher homologues are also present. 3. Strict aerobes are of two types, those that contain 2-polyprenylphenols, 6-methoxy-2-polyprenylphenols and ubiquinones, and those that contain ubiquinones only. The principal homologues are generally octa- or nona-prenyl, although one organism (Agrobacterium tumefaciens) has ubiquinone-10 as its principal homologue. As in the enterobacteria, lower homologues of these compounds are also present. 4. In Escherichia coli W, Pseudomonas ovalis Chester and Pseudomonas fluorescens, radioactivity from p-hydroxy[U-(14)C]benzoic acid is incorporated into 2-polyprenylphenols, 6-methoxy-2-polyprenylphenols, 6-methoxy-3-methyl-2-polyprenyl-1,4-benzoquinones, ubiquinones and a compound tentatively identified as 2-polyprenyl-1,4-benzoquinone. The fact that radioactivity is incorporated into the first three compounds suggests that in these organisms, and indeed in all those Gram-negative bacteria that contain 2-polyprenylphenols and 6-methoxy-2-polyprenylphenols, ubiquinones are formed by a biosynthetic sequence similar to that in Rhodospirillum rubrum. 5. The finding in ;Vibrio O1' (Moraxella sp.) and organism PC4 that 2-polyprenylphenols and 6-methoxy-2-polyprenylphenols are chemically and radiochemically undetectable leads to the conclusion that they are not intermediates in the biosynthesis of ubiquinone by these and by other Gram-negative bacteria that do not contain detectable amounts of 2-polyprenylphenols and 6-methoxy-2-polyprenylphenols. However, ;Vibrio O1' (organism PC4 was not examined) does contain 6-methoxy-3-methyl-2-polyprenyl-1,4-benzoquinone. 6. In Ps. ovalis Chester, radioactivity from l-[Me-(14)C]methionine is incorporated into the nuclear C-methyl and O-methyl groups of 6-methoxy-3-methyl-2-polyprenyl-1,4-benzoquinones and ubiquinone-9, and into the O-methyl group of 6-methoxy-2-polyprenylphenols.

Biosynthesis of phytoquinones. Biosynthetic origins of the nuclei and satellite methyl groups of plastoquinone, tocopherols and tocopherolquinones in maize shoots, bean shoots and ivy leaves.

1. By using dl-[ring-(14)C]phenylalanine, dl-[beta-(14)C]phenylalanine, dl-[alpha-(14)C]-tyrosine and dl-[beta-(14)C]tyrosine it was shown that in maize shoots (Zea mays) the nucleus and one nuclear methyl group of each of the following compounds, plastoquinone, gamma-tocopherol (aromatic nucleus) and alpha-tocopherolquinone, are formed from the nuclear carbon atoms and beta-carbon atom respectively of either exogenous phenylalanine or exogenous tyrosine. With ubiquinone only the aromatic ring of the amino acid is used in the synthesis of the quinone nucleus. Chemical degradation of plastoquinone and gamma-tocopherol molecules labelled from l-[U-(14)C]tyrosine established that a C(6)-C(1) unit directly derived from the amino acid is involved in the synthesis of these compounds. Radioactivity from [beta-(14)C]cinnamic acid is not incorporated into plastoquinone, tocopherols or tocopherolquinones, demonstrating that the C(6)-C(1) unit is not formed from any of the C(6)-C(1) phenolic acids associated with the metabolism of this compound. 2. The incorporation of radioactivity from l-[U-(14)C]tyrosine, dl-[beta-(14)C]tyrosine and dl-[U-(14)C]phenylalanine into bean shoots (Phaseolus vulgaris) and dl-[beta-(14)C]tyrosine and l-[Me-(14)C]methionine into ivy leaves (Hedera helix) was also investigated. Similar results were obtained to those reported for maize, except that in beans phenylalanine is only used for ubiquinone biosynthesis. This is attributed to the absence of phenylalanine hydroxylase from these tissues. In ivy leaves it is found that the beta-carbon atom of tyrosine gives rise to the 8-methyl group of delta-tocopherol, and it is suggested that for all other compounds examined it will give rise to the nuclear methyl group meta to the polyprenyl unit. 3. Preliminary investigations with the alga Euglena gracilis showed that in this organism ring-opening of tyrosine occurs to such an extent that the incorporation data from radiochemical experiments are meaningless. 4. The above results, coupled with previous observations, are interpreted as showing that in higher plants the nucleus of ubiquinone can be formed from either phenylalanine or tyrosine by a pathway involving as intermediates p-coumaric acid and p-hydroxybenzoic acid. Plastoquinone, tocopherols and alpha-tocopherolquinone are formed from p-hydroxyphenylpyruvate by a pathway in which the aromatic ring and C-3 of the side chain give rise respectively to the nucleus and to one nuclear methyl group. 5. Dilution experiments provided evidence that in maize shoots p-hydroxyphenylpyruvic acid and homogentisic acid (produced from p-hydroxyphenylpyruvic acid) are involved in plastoquinone biosynthesis, and presumably the biosynthesis of related compounds: however, other possible intermediates in the conversion including toluquinol (the aglycone of the proposed key intermediate) showed no dilution effects. Further, radioactivity from [Me-(14)C]toluquinol is not incorporated into any of the compounds examined. 6. Dilution experiments with 3,4-dihydroxybenzaldehyde and radioactive-labelling experiments with 3,4-dihydroxy[U-(14)C]benzoic acid demonstrated that these compounds are not involved in the biosynthesis of either ubiquinone or phylloquinone in maize shoots. 7. Evidence is also presented to show that in maize shoots ring-opening of the aromatic amino acids takes place. The suggestion is offered that this may take place via homogentisic acid, as in animals and some micro-organisms.

Biosynthesis of phytoquinones. Incorporation of L-[Me-14C,3H]methionine into terpenoid quinones and chromanols in maize shoots.

1. Radioactivity from l-[Me-(14)C,(3)H]methionine is incorporated into phylloquinone, plastoquinone, gamma-tocopherol, alpha-tocopherol, alpha-tocopherolquinone and ubiquinone in maize shoots. 2. Comparative studies with other terpenoids (squalene and beta-carotene) and chemical degradation of selected quinones (ubiquinone and plastoquinone) established that all the radioactivity is confined to nuclear methyl substituents. 3. In ubiquinone 76% of the radioactivity is in the methoxyl groups and 24% in the ring C-methyl group. 4. Taking the phytosterols as an internal reference and accepting the atomic ratio of (14)C/(3)H transferred from l-[Me-(14)C,(3)H]methionine to the supernumerary group at C(24) to be 1:2 the ratio of all the quinones and chromanols examined approached 1:3. After allowing for the fact that for plastoquinone, gamma-tocopherol, alpha-tocopherol and alpha-tocopherolquinone one nuclear methyl group is formed from the beta-carbon of tyrosine, these results show that one nuclear C-methyl group for phylloquinone, plastoquinone and gamma-tocopherol, two nuclear methyl groups for alpha-tocopherol and alpha-tocopherolquinone and one nuclear methyl and two methoxyl groups for ubiquinone are formed by the transfer of intact methyl groups from methionine. 5. From a comparison of the incorporation of (14)C radioactivity into these compounds it would appear that the methylation reactions involved in phylloquinone and plastoquinone biosynthesis take place in the chloroplast, whereas those involved with ubiquinone biosynthesis occur else-where within the cell.

Observations on the biosynthesis of phytoterpenoid quinone and chromanol nuclei.

1. p-Hydroxy[U-(14)C]benzoic acid, except for loss of the carboxyl group, is effectively incorporated into the nucleus of ubiquinone and an unidentified prenylphenol by maize roots, maize shoots, french-bean leaves, french-bean cotyledons and Ochromonas danica. Plastoquinone, alpha-tocopherol, gamma-tocopherol and alpha-tocopherolquinone are all unlabelled from this substrate. The high radioactivity of the prenylphenol and its behaviour in a pulse-labelling experiment with maize shoots suggested that it may be a ubiquinone precursor. 2. Members of the 2-polyprenylphenol and 6-methoxy-2-polyprenylphenol series, compounds that are known ubiquinone precursors in Rhodospirillum rubrum, could not be detected in maize tissues, but possibly they may occur as their glycosides. 3. [G-(14)C]Shikimic acid is incorporated into the nuclei of phylloquinone, plastoquinone, alpha-tocopherolquinone, gamma-tocopherol, alpha-tocopherol and ubiquinone in maize shoots, showing that in plant tissues the nuclei of these compounds arise via the shikimic acid pathway of aromatic biosynthesis. 4. l-[U-(14)C]Phenylalanine and l-[U-(14)C]tyrosine are incorporated into plastoquinone, gamma-tocopherol, alpha-tocopherolquinone and ubiquinone. alpha-Tocopherol, which is absent from shoots incubated with l-[U-(14)C]tyrosine, is also labelled from l-[U-(14)C]phenylalanine. Degradation studies showed that there is little (14)C radioactivity in the terpenoid portions of the molecules and from this it is concluded that the aromatic portions of these amino acids are giving rise to the quinone and chromanol nuclei. 5. It is proposed that in maize the nucleus of ubiquinone can be formed from either phenylalanine or tyrosine by a pathway involving p-coumaric acid and p-hydroxybenzoic acid. Plastoquinone, tocopherols and tocopherolquinones are formed from tyrosine by some pathway in which the aromatic ring and C-3 of the side chain of this amino acid gives rise to the nucleus and one methyl substituent respectively of these compounds.