Coproporphyrin excretion and low thiol levels caused by point mutation in the Rhodobacter sphaeroides S-adenosylmethionine synthetase gene

Abstract

A spontaneous mutant of Rhodobacter sphaeroides f. sp. denitrificans IL-106 was found to excrete a large amount of a red compound identified as coproporphyrin III, an intermediate in bacteriochlorophyll and heme synthesis. The mutant, named PORF, is able to grow under phototrophic conditions but has low levels of intracellular cysteine and glutathione and overexpresses the cysteine synthase CysK. The expression of molybdoenzymes such as dimethyl sulfoxide (DMSO) and nitrate reductases is also affected under certain growth conditions. Excretion of coproporphyrin and overexpression of CysK are not directly related but were both found to be consequences of a diminished synthesis of the key metabolite S-adenosylmethionine (SAM). The wild-type phenotype is restored when the gene metK encoding SAM synthetase is supplied in trans. The metK gene in the mutant strain has a mutation leading to a single amino acid change (H145Y) in the encoded protein. This point mutation is responsible for a 70% decrease in intracellular SAM content which probably affects the activities of numerous SAM-dependent enzymes such as coproporphyrinogen oxidase (HemN); uroporphyrinogen III methyltransferase (CobA), which is involved in siroheme synthesis; and molybdenum cofactor biosynthesis protein A (MoaA). We propose a model showing that the attenuation of the activities of SAM-dependent enzymes in the mutant could be responsible for the coproporphyrin excretion, the low cysteine and glutathione contents, and the decrease in DMSO and nitrate reductase activities.

FIG. 1.

Tetrapyrrole biosynthetic pathway. Only the names of the intermediates and the genes encoding the enzymes of the pathway are shown. The gene encoding protoporphyrinogen IX oxidase has not been identified in R. sphaeroides 2.4.1 (46).

FIG. 2.

(A) R. sphaeroides f. sp. denitrificans IL-106 wild-type and PORF mutant cultures grown in Sistrom medium under phototrophic conditions (180 μmol photons m⁻² s⁻¹). (B) Absorption spectrum of cell-free supernatant (100-fold dilution) of PORF mutant culture. Inset, magnification of the 460- to 630-nm region of the spectrum.

FIG. 3.

HPLC elution profiles of coproporphyrin I (A), coproporphyrin III (B), and cell-free supernatant of PORF mutant culture grown in Sistrom medium under phototrophic conditions (180 μmol photons m⁻² s⁻¹) (C).

FIG. 4.

Growth curves (A, C, and E) and absorption spectra (B, D, and F) of wild-type R. sphaeroides f. sp. denitrificans IL-106 (blue) and the PORF mutant (pink) under different growth conditions. Phototrophic conditions at 180 μmol photons m⁻² s⁻¹ (A and B), phototrophic conditions at 17 μmol photons m⁻² s⁻¹ (C and D), and dark anaerobic conditions with 60 mM DMSO (E and F) were used. For panels B, D, and F, cultures (in exponential phase) were diluted before measurement to have the same optical density at 660 nm (OD₆₆₀).

FIG. 5.

Two-dimensional PAGE of cytoplasmic extracts (200 μg) of wild-type R. sphaeroides (A) and the PORF mutant (B) grown in Sistrom minimal medium under phototrophic conditions. The arrow shows the cysteine synthase encoded by a homologue of RSP_1109 which is overexpressed in the PORF mutant.

FIG. 6.

Cysteine (A) and glutathione (B) contents of wild-type R. sphaeroides, the PORF mutant, and the PORF mutant with the metK gene in trans during the exponential phase (light gray bars) or the stationary phase (dark gray bars). Monobromobimane derivatives of soluble thiols were separated by reverse-phase HPLC and quantified by comparison with cysteine and glutathione standards. The results are the averages and standard deviations from three independent experiments.

FIG. 7.

Multiple alignment of the SAM synthetases (MetK) of wild-type R. sphaeroides f. sp. denitrificans IL-106 (MetK WT), R. sphaeroides f. sp. denitrificans IL-106 mutant PORF (MetK PORF), and mutants MS737 and MS794 (MetK MS737 and MetK MS794) and of E. coli K-12. Identical residues are shaded gray. Boxes indicate the residues mutated in the mutants which excrete coproporphyrin III.

FIG. 8.

SAM contents of wild-type R. sphaeroides, the PORF mutant, and the PORF mutant containing pMS931 (with metK) in trans. Deproteinated soluble cell extracts were separated by reverse-phase HPLC and quantified by comparison with SAM standards. The results are the averages and standard deviations from three independent experiments.

FIG. 9.

Effects of a decrease in SAM-dependent enzyme activity on tetrapyrrole and cysteine synthesis pathways. For clarity, only the names of enzymes whose activities are modified are shown. A drop in SAM concentration would decrease the activities of two SAM-dependent enzymes, coproporphyrinogen oxidases (HemN/Z) and uroporphyrinogen methylase (CobA). This would result in the accumulation of coproporphyrinogen III and a decrease in siroheme synthesis. Activity of the sulfite reductase (CysI) would be affected by the shortage of siroheme; the concentration of sulfide, and consequently also that of cysteine, would decrease. Accumulation of O-acetylserine (OAS) would result in the induction of the cysteine regulon with overexpression of CysK. Cofactors whose synthesis is affected are shown in brackets. Compounds whose concentration decreases are shown in gray. Compounds or enzymes whose concentration increases are underlined.