Proc. Natl. Acad. Sci. USA Vol. 95, pp. 4853–4858, April 1998 Biochemistry A primitive pathway of porphyrin biosynthesis and enzymology in Desulfovibrio vulgaris TETSUO ISHIDA*, LING YU*, HIDEO AKUTSU†,KIYOSHI OZAWA†,SHOSUKE KAWANISHI‡,AKIRA SETO§, i TOSHIRO INUBUSHI¶, AND SEIYO SANO* Departments of *Biochemistry and §Microbiology and ¶Division of Biophysics, Molecular Neurobiology Research Center, Shiga University of Medical Science, Seta, Ohtsu, Shiga 520-21, Japan; †Department of Bioengineering, Faculty of Engineering, Yokohama National University, 156 Tokiwadai, Hodogaya-ku, Yokohama 240, Japan; and ‡Department of Public Health, Graduate School of Medicine, Kyoto University, Sakyou-ku, Kyoto 606, Japan Communicated by Rudi Schmid, University of California, San Francisco, CA, February 23, 1998 (received for review March 15, 1998) ABSTRACT Culture of Desulfovibrio vulgaris in a medium billion years ago (3). Therefore, it is important to establish the supplemented with 5-aminolevulinic acid and L-methionine- biosynthetic pathway of porphyrins in D. vulgaris, not only methyl-d3 resulted in the formation of porphyrins (sirohydro- from the biochemical point of view, but also from the view- chlorin, coproporphyrin III, and protoporphyrin IX) in which point of molecular evolution. In this paper, we describe a the methyl groups at the C-2 and C-7 positions were deuter- sequence of intermediates in the conversion of uroporphy- ated. A previously unknown hexacarboxylic acid was also rinogen III to coproporphyrinogen III and their stepwise isolated, and its structure was determined to be 12,18- enzymic conversion. didecarboxysirohydrochlorin by mass spectrometry and 1H NMR. These results indicate a primitive pathway of heme biosynthesis in D. vulgaris consisting of the following enzy- MATERIALS AND METHODS matic steps: (i) methylation of the C-2 and C-7 positions of Materials. Uroporphyrin III octamethyl ester, copropor- uroporphyrinogen III to form precorrin-2 (dihydrosirohydro- phyrin III tetramethyl ester, and protoporphyrin IX dimethyl chlorin); (ii) decarboxylation of acetate groups at the C-12 ester were obtained from Sigma. ALA was obtained from and C-18 positions of precorrin-2 to form 12,18-didecar- Wako Pure Chemical (Kyoto). Deuterated L-methionine- boxyprecorrin-2; (iii) elimination of acetate groups of the C-2 methyl-d3 was purchased from Cambridge Isotope Laborato- and C-7 positions of 12,18-didecarboxyprecorrin-2 to form ries (Cambridge, MA). coproporphyrinogen III; and (iv) conversion of coproporphy- Culture Conditions. D. vulgaris Miyazaki F and Hildenbor- rinogen III to protoporphyrin IX via protoporphyrinogen IX. ough cells were cultured anaerobically on Postgate medium C We isolated the following three enzymatic activities involved in (4) containing ALA (10 mgyliter) and L-methionine (0.2 steps i–iii from the soluble fraction of the cells by anion- gyliter) for 18 h at 37°C. exchange chromatography: S-adenosyl-L-methionine:uropor- Isolation and Characterization of Porphyrins. The bacterial phyrinogen III methyltransferase, precorrin-2 12,18-acetate cells were collected by centrifuging the culture medium at decarboxylase, and 12,18-didecarboxyprecorrin-2 2,7- 11,000 3 g. The pellets were resuspended in 5 vol of l0 mM decarboxymethylase; all enzymic products were converted TriszHCl, pH 7.3, containing 5 mM dithiothreitol (DTT). The into autooxidized methyl esters and analyzed by thin-layer chromatography, UV–visible (UV-VIS) absorption, and mass mixture was sonicated with 10–15 1-min bursts at 0°C. After DNase I (Sigma) was added, the mixture was centrifuged at spectrometry. The enzymatic reactions in D. vulgaris shed new 3 light on porphyrin biosynthesis at an early stage in the 45,000 g for1hat4°C. The supernatant and the pellet were evolution of prokaryotes. regarded as the soluble and cell membrane fractions, respec- tively. Porphyrins were extracted from the soluble and the mem- Porphyrin biosynthesis in aerobic organisms has been exten- brane fractions with 10–20 vol of 0.1 M HClyacetone and 0.5 sively investigated and the pathway is well established (1). M HClyacetone, respectively, according to a method described Although the source of 5-aminolevulinic acid (ALA) can be previously (5, 6). The HClyacetone solution was evaporated to either glycine plus succinyl-CoA or glutamate, depending on the species, the biosynthetic pathway from ALA to protopor- dryness under reduced pressure, and the solid was subjected to phyrin IX is common to all aerobic organisms so far examined. methyl esterification. An efficient way to prepare methyl esters of siro- and siro-type hydrochlorin without formation of However, Akutsu, Park, and Sano discovered in 1993 that the m methyl groups at the C-2 and C-7 positions of heme c in lactone is as follows. The extracted porphyrins (about 1 mol) were kept in one arm of a Thunberg-type tube and anhydrous cytochrome c3 from the obligate anaerobe Desulfovibrio vul- garis Miyazaki F arise, not from C-2 of ALA, as in the HCl in methanol (5–7%, 5–7 ml) containing 50 mg of pow- established pathway, but from the methyl group of L- dered anhydrous FeSO4 was kept in the other arm of the tube. methionine (2). This finding suggested that an alternative The system was flushed slowly with nitrogen and the contents pathway from uroporphyrinogen III to protoheme operates in were mixed, followed by the passage of nitrogen gas into the this sulfate-reducing bacterium. solution at a very fast flow for 2 min at 50°C. After the tube Sulfate-reducing bacteria belonging to the genus Desulfo- was closed, the mixture was kept overnight at room temper- vibrio are included among the domain eubacteria and are ature. The mixture was then neutralized with saturated sodium considered to be closely related phylogenetically to the earliest acetate and the ester was extracted into ethyl acetate, washed living organisms, which appeared on earth approximately 3 Abbreviations: ALA, 5-aminolevulinic acid; LDIMS, laser desorption The publication costs of this article were defrayed in part by page charge ionization mass spectrometry; LSIMS, liquid secondary ion mass spectrometry; UV-VIS, UV-visible; SAM, S-adenosyl-L-methionine; payment. This article must therefore be hereby marked ‘‘advertisement’’ in SUMT, S-adenosyl-L-methionine:uroporphyrinogen III methyltrans- accordance with 18 U.S.C. §1734 solely to indicate this fact. ferase. i © 1998 by The National Academy of Sciences 0027-8424y98y954853-6$2.00y0 To whom reprint requests should be addressed. e-mail: ssano@mbox. PNAS is available online at http:yywww.pnas.org. kyoto-inet.or.jp. 4853 Downloaded by guest on September 30, 2021 4854 Biochemistry: Ishida et al. Proc. Natl. Acad. Sci. USA 95 (1998) with water, dried over sodium sulfate, and evaporated to ylase activity was measured in a final volume of 2 ml containing dryness under reduced pressure. 0.1 M TriszHCl buffer at pH 7.7, 0.1 M NaCl, 5 mM DTT, 0.5 For purification of porphyrin esters, preparative TLC was mM S-adenosyl-L-methionine (SAM), 5 mM uroporphyrino- performed on silica gel plates (Kieselgel 60, Merck) with gen III [prepared by the reduction of uroporphyrin III with benzeneyethyl acetateymethanol (85:12:3, volyvol) as an sodium amalgam (7)], and the enzyme solution. The same eluent. The methyl esters of porphyrins were also separated by experiment was done in the absence of SAM. The mixture was HPLC using a YMC porous silica column (s-5 120A SIL, incubated anaerobically in the dark at 37°C for 1–3 h, and the Yamamura Chemical Laboratories, Kyoto; 2 3 25 cm) or a reaction was stopped by the addition of 20 ml of HClyacetone Cosmosil 5SL column (Nacalai Tesque, Kyoto; 4.6 3 150 mm). (0.5 M) at 0°C. After being centrifuged, the supernatant was The various porphyrin esters were eluted with n-heptaneyethyl completely dried under reduced pressure. The extracted por- acetateydichloromethaneymethanol (60:25:15:2.7, volyvol) phyrins were subjected to methyl esterification and separated and were detected by monitoring fluorescence at 600 nm with by TLC. SUMT activity was determined by the amount of excitation at 380 nm (siro- or siro-type porphyrins) or fluo- sirohydrochlorin octamethyl ester recovered from the reaction rescence at 620 nm with excitation at 400 nm (uroporphyrin, mixtures. coproporphyrin, protoporphyrin, etc.) by means of a Shimadzu (ii) Precorrin-2 decarboxylase assay. Precorrin-2 decarboxyl- RF-535 fluorescence detector. ase catalyzes the conversion of precorrin-2 to 12,18- Absorption spectra were recorded by a Shimadzu UV- didecarboxyprecorrin-2. Because precorrin-2 is extremely sen- 3100PC UV-VIS-NIR scanning spectrophotometer or a Shi- sitive to oxidation and the reduction of sirohydrochlorin with madzu UV-2200 spectrophotometer. Laser desorption ioniza- sodium amalgam resulted in a compound different from tion mass spectrometry (LDIMS) was performed on a Shi- precorrin-2, the precorrin-2 was generated from uroporphy- madzuyKratos Kompact MALDI III laser ionization time-of- rinogen III and SAM by using partially purified SUMT. Thus, flight mass spectrometer equipped with a nitrogen laser (337 the decarboxylase activity was anaerobically measured in a nm). Ions were accelerated to a kinetic energy of 5 keV and final volume of 2 ml containing 0.1 M TriszHCl at pH 7.7, 0.1 were analyzed in the positive linear mode of operation. The M NaCl, 5 mM DTT, 0.5 mM SAM, 0.5 mM NADH, 0.5 mM laser power density was 106 Wzcm22. High-resolution liquid NADPH, 5 mM uroporphyrinogen III, partially purified secondary ion mass spectrometry (LSIMS) was performed SUMT, and the enzyme solution. As a control, the same with a ShimadzuyKratos Concept I.H. instrument. 1H NMR experiment was done in the absence of SAM. After incubation, spectra were obtained with a JEOL JNM-GX-270 NMR the porphyrins were extracted and subjected to methyl ester- spectrometer. ification as described for SUMT assay. The decarboxylase Preparation of Enzymes.