Biosynthesis of Caldariellaquinone in Sulfolobus acidocaldarius

The biosynthesis of caldariellaquinone (CQ) has been studied in Sulfolobus acidocaldarius using a variety of methods. By growing cells with a series of tyrosines labeled with deuterium or ¹³C and measuring the extent and position at which label was incorporated into the CQ by mass spectrometry, it was concluded that the benzo[b]thiophen-4,7-quinone ring of CQ is derived as an intact unit from all of the carbons of tyrosine except C-1. Additional work, using (3S)-L-(2-²H, 3-²H]-, (3R)-D-[2-²H, 3-²H]-, (3S)-D-[3-²H]-, and (3R)-L-[3-²H]- tyrosine, demonstrated that the pro-3S hydrogen of either D- or L-tyrosine is the origin of the C-3 proton of the benzo[b]thiophene ring.

Considering the above information and the structure of CQ, it was concluded that CQ was most likely biosynthesized by the condensation of farnesylfarnesyl pyrophosphate with homogentisic acid (HA) in a reaction analogous to that found in the biosynthesis of ubiquinone. The possibility of this reaction being involved in the biosynthesis of CQ was supported by the identification of farnesylfarnesol, a hydrolytic breakdown product of farnesylfarnesyl pyrophosphate, by gas chromatography-mass spectrometry (GC-MS) of purified lipid extracts. The possible involvement of HA in CQ biosynthesis, however, could not be confirmed by five independent methods. The possible formation of CQ by the condensation of benzo[b]thiophen-4,7-quinone with farnesylfarnesyl pyrophosphate was eliminated by the inability to detect benzo[b]thiophen-4,7-quinone in S. acidocaldarius.

Attempts to identify the tyrosine metabolites leading to CQ by studing the metabolisms of tyrosine, 2-fluorotyrosine, and 3-fluorotyrosine in S. acidocaldarius lead to the identification of two previously undescribed pathways for tyrosine metabolism. These two pathways branch after the conversion of tyrosine to 4-hydroxyphenylacetic acid (pHPA). The ability of labeled pDHPA to be incorporated into these metabolites, but not into CQ, indicates that the first committed step in the biosynthesis of CQ occurs at either tyrosine or a metabolite very closely related to tyrosine, e.g., 4-hydroxyphenylpyruvate (pHPP). Analysis of the extract of the cells grown with 3-fluorotyrosine showed two fluorine-containing compounds, which are likely to be fluoro-analogues of the intermediates in the biosynthesis of CQ. However, because of the small amount of these two compounds found (24 nmoles/g of wet weight), structural characterization was not possible.

Both the methyl and sulfur groups of the methylthio portion of CQ were shown to arise from methionine. Mass spectral analysis of the CQ isolated from cells grown in the presence of [³⁴S-methyl-²H₃]-L-methionine clearly showed, however, that the methylthio group of CQ is not derived as an intact unit from the methylthio group of methionine. Additional work supported the theory that the methionine sulfur first undergoes transsulfuration to cysteine, which then supplies the sulfur for both the methylthio and the benzo[b]thiophene moieties of CQ. This represents the first example of transsulfuration from methionine to cysteine occurring in archaebacteria.