(Received for publication, December 26, 1995; and in revised form, January 23, 1996)
From the
Plasmids containing DNA from the green photosynthetic bacterium Chlorobium vibrioforme complement a heme-requiring Escherichia coli hemB mutant that is deficient in
porphobilinogen (PBG) synthase activity. PBG synthase activity was
detected in extract of complemented cells but not in that of cells
transformed with control plasmid. The sequence of the C.
vibrioforme hemB gene predicts a HemB protein that contains 328
amino acids, has a molecular weight of 36,407, and is 53% identical to
the homologous proteins of Synechocystis sp. PCC 6301 and Rhodobacter capsulatus. The response of C. vibrioforme PBG synthase to divalent metals is unlike that of any previously
described PBG synthase; Mg stimulates but is not
required for activity, and Zn
neither stimulates nor
is required. This response correlates with predicted sequences of two
putative variable metal binding regions of C. vibrioforme HemB. The C. vibrioforme hemB open reading frame begins
1585 bases downstream from the end of the hemD open reading
frame and is transcribed in the same direction as hemA, hemC, and hemD. However, hemB is not part of
the same transcription unit as these genes, and the hemB transcript is approximately the same size as the hemB gene alone. Between hemD and hemB there is an
intervening open reading frame that is oriented in the opposite
direction and encodes a protein with a predicted amino acid sequence
significantly similar to that of inositol monophosphatase, an enzyme
that is not involved in tetrapyrrole biosynthesis. The gene order
within hem gene clusters is highly conserved in
phylogenetically diverse prokaryotic organisms. This conservation
suggests that there are functional constraints on the relative order of
the hem genes.
Biosynthesis of porphyrins and related compounds proceeds via a
common set of intermediates from ALA, ()the first universal
precursor, through the first cyclic tetrapyrrole, uroporphyrinogen III,
at which point the pathway splits into two branches, one leading to
reduced products such as siroheme and vitamin B
and the
other leading to oxidized end products, including hemes, bilins,
chlorophylls, and bacteriochlorophylls (Fig. 1). There are two
routes to ALA, one that involves the condensation of glycine and
succinyl-coenzyme A and occurs exclusively in
-proteobacteria and
nonphototrophic eukaryotes and the other more common pathway that
begins with glutamyl-tRNA (Beale, 1995).
Figure 1: Early steps of the tetrapyrrole biosynthetic pathway with the gene products under discussion indicated for the reactions they catalyze.
Although the early biosynthetic steps from ALA onward are identical in all species examined, there are some interesting differences in the properties of certain enzymes from different sources. For example, PBG synthase (also known as ALA dehydratase) obtained from different species has different metal requirements for activity (Jaffe, 1993, 1995). Another difference is that in some species, the cysG gene product, siroheme synthase, catalyzes three sequential steps in the conversion of uroporphyrinogen III to siroheme, whereas in other species, a different enzyme, S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase, catalyzes only the first step, forming precorrin-2 (Spencer et al., 1993; Warren et al., 1994). Interestingly, the amino acid sequence of the methyltransferase is similar to that of the C-terminal portion of siroheme synthase (Spencer et al., 1993).
Genes for the enzymes catalyzing the early steps of tetrapyrrole biosynthesis have been cloned and sequenced from several prokaryotic species. Often, two or more of the genes are arranged in a cluster in the genome, and in some cases the clustered genes comprise a common transcription unit (Hansson et al., 1991; Jordan et al., 1988). There is wide variation among species in the identity and number of clustered hem genes, and this variation may have both regulatory significance and evolutionary implications.
Chlorobium vibrioforme is a strictly anaerobic green phototrophic bacterium. Green bacteria have been very useful for comparative and evolutionary studies of photosynthesis and related processes because they are only distantly related to other photosynthetic organisms; their photosynthetic reaction center and light harvesting apparatus are completely different from those of the other group of phototrophic anaerobes, the purple bacteria, and their mode of carbon fixation is totally unlike that of purple bacteria, cyanobacteria, or plants (Blankenship et al., 1995; Feiler and Hauska, 1995; Sirevåg, 1995). Previous studies of C. vibrioforme provided the first information about the structure of tetrapyrrole biosynthetic enzymes and their genes in any strict anaerobe. C. vibrioforme has a gene cluster that contains three hem genes, hemA, hemC, and hemD, which encode glutamyl-tRNA reductase, hydroxymethylbilane synthase, and uroporphyrinogen III synthase, respectively (Majumdar et al., 1991; Moberg and Avissar, 1994). These three genes appear to comprise an operon that yields a transcript of sufficient size to encompass all three open reading frames (Majumdar et al., 1991).
We now communicate the sequence of the C. vibrioforme hemB gene, describe its positional relationship to and expressional independence from the hemACD genes, and report on some catalytic properties of its encoded enzyme, PBG synthase.
DNA sequencing of the 2.8-kilobase insert of pYA4 was done in both directions using an fmol DNA sequencing system kit (Promega, Madison, Wisc.) according to the manufacturer's protocol. Sequence information was compiled with the MacVector DNA sequence analysis program (Eastman Kodak Co.).
As a probe for Northern hybridizations, a 827-bp fragment of
pYA4 containing most of the C. vibrioforme hemB coding region
was prepared by polymerase chain reaction using the oligonucleotides
5`-GCATCGCCCGAGAAG-3` and 5`-TCACCATGGCGTATTCG-3` for the sense and
antisense primers, respectively. The 827-bp fragment was purified by
electrophoresis in low melting point agarose followed by elution and
phenol extraction and labeled with [P]dATP by
the nick translation method using a kit obtained from Life
Technologies, Inc.
The standard
incubation medium contained 100 mM Bis-Tris-Propane-HCl, pH
8.5, 1 mM -mercaptoethanol, 10 mM ALA, 50 mM KCl, 10 mM MgCl
, and cell extract (additions
and variations are described under ``Results and
Discussion''). Incubation was for 15 or 30 min at 32 °C in a
total volume of 0.5 ml. Incubations were stopped by the addition of 0.5
ml of modified Ehrlich-Hg reagent (Urata and Granick, 1963) and mixing.
The mixture was clarified by centrifugation for 2 min in the
microcentrifuge, and the absorbance was recorded at 555 nm between 5
and 15 min after the addition of the modified Ehrlich-Hg reagent. The
PBG concentration was calculated using a molar absorption value of
68,000 (Urata and Granick, 1963).
To
verify that the complementation was caused by the expression of hemB, complemented RP523 cells were examined for the presence
of PBG synthase activity. This verification was necessary because in
some cases pseudo-complementation can be caused by overexpression of
other hem genes. For example, overexpression of C.
vibrioforme hemA in an E. coli hemL strain leads to
heme-independent growth. ()This pseudo-complementation
presumably results from nonenzymatic conversion of
glutamate-1-semialdehyde to ALA, which can occur at high
glutamate-1-semialdehyde concentrations (Hoober et al., 1988).
Extracts of RP523 cells that were complemented with pYA4 had PBG
synthase levels comparable with those of the hemB parental strain C600 (Table 2). In contrast, extracts of
RP523 cells that were complemented with the pBluescript SK(+)
vector were devoid of PBG synthase activity. Because it was necessary
to add heme to the medium of uncomplemented RP523 cells to obtain
growth, it was possible that the absence of PBG synthase in RP523 cell
extract was due to repression of its formation or inhibition of its
activity caused by the added heme. Therefore, PBG synthase activity was
determined in extracts of RP523 cells that were transformed with pYA4
but grown in the presence of added heme. These cells had levels of PBG
synthase activity equal to that of complemented cells grown without
added heme. We therefore conclude that the complete absence of PBG
synthase activity in uncomplemented RP523 cells is not caused by the
heme added to the medium and that the activity in RP523 cells
complemented by pYA4 is attributable to the expression of C.
vibrioforme hemB in the transformed cells.
In the previous complementation studies with C. vibrioforme hemA, hemC, and hemD (Avissar and Beale, 1990; Moberg and Avissar, 1994), there was some uncertainty about whether the transcription of these genes in E. coli cells was directed by promoter elements on the C. vibrioforme DNA or from the lac promoter on the pBluescript SK(+) vector. For hemB, the size of the transcript (see below) indicates that transcription begins well within the inserted DNA, approximately 1750 bp from the beginning of the inserted C. vibrioforme DNA (Fig. 2). Moreover, there is a transcription stop signal between the end of the hemD gene and the beginning of the hemB gene. Therefore, it is likely that the C. vibrioforme promoter for the hemB gene can function in E. coli sufficiently well to cause complementation and produce measurable PBG synthase activity in cell extracts.
Figure 2: Nucleotide sequence of a 2872-bp cloned C. vibrioforme genomic DNA insert in pYA4, the deduced peptide sequence for the C-terminal 60 amino acids of the hemD gene product, and the deduced peptide sequence for the complete 328-amino acid hemB gene product. Stop codons are indicated by asterisks. For hemB, the -10 (Pribnow box) consensus sequence is indicated by asterisks, and a potential -35 region consensus sequence is indicated by double underlining. For HemB, putative metal binding sites B (residues 122-140) and C (residues 228-238) and the active site lysine (residue 253) are indicated with single underlining. Putative rho-independent transcription termination sequences downstream from the ends of hemD and hemB are shown in bold letters with the palindromic regions indicated by arrowheads. The GenBank accession number is U38348.
The deduced C. vibrioforme HemB sequence is significantly similar to all published PBG synthase sequences. The most similar sequence in the GenBank data base is that of PBG synthase from the cyanobacterium Synechocystis sp. PCC 6301 (Jones et al., 1994), which is 53.0% identical to C. vibrioforme HemB (Fig. 3). Even the least similar PBG synthase, the human enzyme (Wetmur et al., 1986), is 36.9% identical to C. vibrioforme HemB. In all cases, the similarity extends throughout the entire polypeptide sequence.
Figure 3: Comparison of the deduced amino acid sequence of C. vibrioforme (C. vib.) HemB with the most similar sequence in the GenBank data base, that of Synechocystis (Syn.) sp. PCC 6301 PBG synthase (GenBank accession number X70434), and the least similar PBG synthase sequence in the GenBank data base, that of the human enzyme (GenBank accession number M13928).
C. vibrioforme PBG synthase
has a broad pH optimum centered at pH 8.5 (Fig. 4). The C.
vibrioforme enzyme appears to represent a new class of PBG
synthase with respect to the effects of divalent metals. The
gel-filtered enzyme does not require the addition of either
Zn or Mg
for activity, but it is
stimulated approximately 2-3-fold (depending on the incubation
pH) by Mg
(Table 3). Zn
is
inhibitory. K
is somewhat stimulatory in the absence
of Mg
, especially at the higher incubation pH, but
K
does not stimulate in incubations containing
Mg
. The absence of inhibition by EDTA reinforces the
conclusion that activity does not require Zn
or
Mg
in the incubation medium.
Figure 4:
pH dependence of C. vibrioforme PBG synthase activity. Gel-filtered C. vibrioforme extract was incubated for 15 min at 32 °C in 0.5 ml of medium
containing 100 mM Bis-Tris-Propane (titrated to the indicated
pH with HCl), 1 mM -mercaptoethanol, 10 mM ALA,
50 mM KCl, 10 mM MgCl
, and cell extract
(43 µg of protein).
In one model that
attempts to relate protein structural features to divalent metal
effects, PBG synthase is proposed to have three metal binding sites
(Jaffe, 1993, 1995). Site A, which is proposed to bind Zn very tightly in all PBG synthases, has not been identified. Site
B (Fig. 5) in the Zn
-requiring enzymes has
several cysteine and histidine residues, which are replaced by
carboxyl-containing residues in the Mg
-requiring
enzymes. Site C has several carboxyl-containing residues in the
Zn
-requiring enzymes that are stimulated by
Mg
, and these residues are absent from the enzymes
that are not stimulated by Mg
. For the
Mg
-requiring enzymes, it is difficult to determine
experimentally whether Mg
additionally stimulates by
binding at site C.
Figure 5: Comparison of the amino acid sequences of putative metal binding regions B and C of PBG synthases from several sources. The enzymes are grouped on the basis of sequence similarity at the B site. For the B site sequences, cysteines, histidines, and residues with carboxyl groups (aspartate and glutamate) are shown in bold letters, and for the C site sequences, residues with carboxyl groups are shown in bold letters. For conserved residues shown in the top line, capital letters are used to indicate absolutely invariant residues, and lowercase letters are used to indicate residues conserved in all but one or two sequences. The peptide sequences for the bovine enzyme are taken from Markham et al.(1993).
The responses of C. vibrioforme PBG
synthase to divalent metals correlate well with the structures of the
putative metal binding sites B and C (Fig. 5). Site B of the C. vibrioforme enzyme most closely resembles that of R.
capsulatus PBG synthase, an enzyme that has no requirements for
divalent metals in the incubation medium. However, site C of the C.
vibrioforme enzyme more closely resembles those of
Zn-requiring PBG synthases that are stimulated by
Mg
. In summary, these results for C. vibrioforme PBG synthase provide strong support for the model identifying
metal binding sites B and C.
PBG synthase from some species has been
reported to be allosterically inhibited by heme, a tetrapyrrole end
product (Nandi et al., 1968). The effect of heme on C.
vibrioforme PBG synthase activity was tested in incubations done
at pH 8.5 in the presence of 50 mM K and 10
mM Mg
. In this experiment, hemin was added
to the incubation mixture from a concentrated stock solution in
dimethyl sulfoxide, and all incubations contained the same dimethyl
sulfoxide concentration (2%, v/v). PBG synthase activity was not
inhibited even by 100 µM heme, the highest concentration
tested (data not shown).
Figure 6: Northern blot of C. vibrioforme total RNA hybridized with an 827-bp hemB-specific probe. RNA size markers were run on a duplicate gel and stained with ethidium bromide. kb, kilobases.
Figure 7: A, nucleotide sequence of a region of the C. vibrioforme DNA in pYA4 for the strand complementary to that which encodes the hemD and hemB genes, and the deduced peptide sequence of its open reading frame. The stop codon is indicated by an asterisk. Potential -10 (Pribnow box) and -35 region consensus sequences are indicated by asterisks and double underlines, respectively. B, comparison of the deduced peptide sequence in A with the amino acid sequences of human (GenBank accession number X66922) and E. coli (GenBank accession number M34828) inositol monophosphatase.
The clustering of hem genes is common in bacteria (Fig. 8) and may have regulatory as well as evolutionary implications. For example, the widespread close clustering and cotranscription of hemC and hemD probably ensures the presence of the hemD product, uroporphyrinogen III synthase, whenever the hemC product, hydroxymethylbilane synthase, is present. The activity of uroporphyrinogen III synthase is necessary to direct the conversion of hydroxymethylbilane to the physiologically relevant product uroporphyrinogen III and prevent its spontaneous conversion to uroporphyrinogen I, a nonphysiological dead-end product. Extreme regulatory coordination is found in B. subtilis, where the genes for all of the enzymes needed for conversion of glutamyl-tRNA to uroporphyrinogen III (Fig. 1), as well as a gene of uncertain function, are clustered on the hemAXCDBL operon (Hansson et al., 1991).
Figure 8: Structures of gene clusters from several prokaryotic species that contain genes for early tetrapyrrole biosynthetic steps. The extent of sequenced regions is indicated by horizontal lines, open reading frames of identified and putative genes are indicated by boxes, and the deduced directions of transcription are indicated by arrows. For ease of comparison, the sequences are arbitrarily aligned at the beginning of the hemC open reading frame, except in the case of the Synechocystis sp. PCC 6301 cluster, which does not contain hemC. In this case, the end of the cobA open reading frame is aligned with the end of the cysG open reading frames of C. josui and Mycobacterium leprae to indicate that the CobA sequence is similar to the C-terminal end of the CysG sequence. The GenBank accession numbers are: C. vibrioforme (M96364, U38348), B. subtilis (M57676), E. coli (X12614), Pseudomonas aeruginosa (M74844), Synechocystis sp. PCC 6301 (X70434), C. josui (D28503), M. leprae (U00018).
From an evolutionary perspective, it is of interest that wherever hem genes are clustered, the sequential arrangement of the genes within the cluster is generally conserved in the order ACDBL (Fig. 8). This arrangement does not follow the order of their products as enzymes catalyzing early steps of the biosynthetic pathway, which is ALBCD (Fig. 1). It is especially interesting that the sequential arrangement of these hem genes has been maintained even when they are not within the same transcription unit and despite the presence of intervening genes between hem genes, such as in C. vibrioforme where a putative inositol monophosphatase-encoding gene is between hemD and hemB and is oriented in the opposite direction. The order of the clustered hem genes is conserved even in the extreme case of Clostridium josui, where there has been apparent fusion of different parts of a split cysG homolog to the hemA and hemD genes (Fujino et al., 1995). The high degree of conservation of the order of clustered hem genes in these phylogenetically diverse organisms strongly suggests that there are functional constraints on their relative genomic position. Critical assessment of this suggestion will require information about the structure of hem gene clusters in additional species.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U38348[GenBank].
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