Center for Biological NMR, Department of Chemistry, Texas A&M University, College Station, TX 77843-3255, USA1
School of Biological Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, UK2
Author for correspondence: Charles A. Roessner. Tel: +1 979 845 3243. Fax: +1 979 845 5992. e-mail: c-roessner{at}tamu.edu
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ABSTRACT |
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Keywords: hem operon, methylmalonyl-CoA mutase, transcarboxylase, metabolic regulation, genetic linkage
The GenBank accession numbers for the sequences reported in this paper are AY033235, AY033236, U13043 and U51164.
a Present address: Protein Engineering, Iogen Corp., 400 Hunt Club Road, Ontario, Ottawa, Canada K1V 1C1.
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INTRODUCTION |
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Note on nomenclature. Since it has been demonstrated that Ps. denitrificans requires O2 for the synthesis of B12 and that S. typhimurium synthesizes B12 only under strict anaerobic conditions, the nomenclatures assigned to the Ps. denitrificans genes (cobAV) and the S. typhimurium genes (cysG, cbiAQ and cobSTU) have become associated with the aerobic and anaerobic pathways to B12, respectively (see Table 1). P. freudenreichii synthesizes B12 anaerobically; therefore the S. typhimurium nomenclature will be used herein as much as possible. Unfortunately, no convention was followed when naming the genes from different organisms, with result that many of the genes encoding the same enzyme have different names whereas some of the genes with the same name encode different enzymes in the two organisms (e.g. cobA, cobB, cobC, cobD, cobS, cobT, cobU). Since two P. freudenreichii cobA genes are described in this manuscript, they will be distinguished by using cobA for the gene encoding uroporphyrinogen III methyltransferase, based on prior usage (Sattler et al., 1995
), and cobAadoT for the gene encoding ATP:corrinoid adenosyltransferase.
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METHODS |
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For SDS-PAGE analysis of the gene products of the cbiXcysGB and cbiEGH genes described herein (Fig. 1), the genes were amplified by PCR providing optimal Escherichia coli translational signals and inserted into expression vectors as previously described (Roessner et al., 1995
). The sequence of the cbiXcysGB forward primer was: CCCGGGGTCGACAAGCTTAGGAATTTAAAATGACTGATCTCGTCCC-ACTGGTCATTGCC, providing a 5' HindIII site (underlined), a ribosome-binding site (italics) and codons for the first 10 amino acids of the protein including the ATG start codon. The sequence of the cbiXcysGB reverse primer was CCCGGGTCTAGAGGATCCTTAGACCTGGAGCGCC-TTGAGGAGCTCGTCGCG, providing a 3' BamHI site (underlined), a stop anticodon (TTA) and anticodons for the last 10 amino acids of the protein. The PCR product was cut with HindIII and BamHI and ligated into pUC19 to give pCR487. The sequence of the cbiEGH forward primer was GGTACCCGGGGATCCAGGAGGAATTTAAAATGATTCGCGTACATGGTTTCCTCGGCGGC, providing a 5' BamHI site (underlined), a ribosome-binding site (italics) and codons for the first 10 amino acids of the protein including the ATG start codon. The sequence of the cbiEGH reverse primer was CGCGCAAGCTTTTAGTCATGGGAATCCTCCTGGGTGGGGACATCGGAGCC, providing a 3' HindIII site (underlined), a stop anticodon (TTA) and anticodons for the last 12 amino acids of the protein. The PCR product was cut with BamHI and HindIII and ligated into pET23a(+) to form pCR571.
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RESULTS AND DISCUSSION |
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The genes necessary for the conversion of precorrin 2 to cobyrinic acid in P. freudenreichii are contained in two convergent operons
In Ps. denitrificans the eight genes required for the aerobic conversion of precorrin 2 to hydrogenobyrinic acid (cobFM) are contiguous (Crouzet et al., 1990 ). Similarly, the genes of S. typhimurium and B. megaterium believed to be necessary for the anaerobic conversion of cobalt-precorrin 2 to cobyrinic acid are found in large B12 operons (Roth et al., 1993
; Raux et al., 1998
). Within an 11·6 kb fragment of P. freudenreichii DNA (Fig. 1b
), we found genes homologous to 10 different genes of S. typhimurium (cysGB, cibC, cbiD, cbiE, cbiF, cbiG, cbiH, cbiJ, cbiL and cbiT) and to one gene of B. megaterium (cbiX). However, since some of the genes are combined into a single larger gene in P. freudenreichii, the two operons encode only eight proteins (see Fig. 1b
and discussed below).
CysG has been proposed to be a multifunctional enzyme for the synthesis of sirohaem in E. coli and S. typhimurium (Spencer et al., 1993 ; Warren et al., 1994
). The C-terminal domain of CysG (CysGA) is a methyltransferase that catalyses the attachment of the first two methyl groups to uroporphyrinogen III to form precorrin 2, whereas the N-terminal domain (CysGB) is believed to catalyse the NADP-dependent dehydrogenation of the macrocycle and iron chelation to give sirohaem. Genetic evidence has been provided (Fazzio & Roth, 1996
; Raux et al., 1998
) that, in S. typhimurium, CysG may also catalyse not only the bismethylation of uroporphyrinogen III but also the insertion of cobalt into precorrin 2 (Fig. 2a
) to give cobalt-precorrin 2, an intermediate unique to the anaerobic vitamin B12 pathway (cobalt insertion occurs at a much later stage in the aerobic pathway into hydrogenobyrinic acid a,c-diamide to form cobyrinic acid a,c-diamide, the structure of which is shown in Fig. 2b
). However, it has also been demonstrated that S. typhimurium has an additional gene, cbiK, that can substitute for cysGB (Raux et al., 1997
) and may encode the actual cobaltochelatase. A gene encoding uroporphyrinogen III methyltransferase was previously isolated from P. freudenreichii (Sattler et al., 1995
) which, in contrast to cysG, contained no dehydrogenase or chelatase (cysGB) encoding region. It was therefore called cobA rather than cysG after the similar gene in Ps. denitrificans. The P. freudenreichii homologue to cysGB is found within a gene whose product is similar not only to cysGB but also to cbiX found in B. megaterium (Raux et al., 1998
). The P. freudenreichii gene encodes a protein of 414 amino acids, the C-terminal 150 of which are similar (32% identity, 50% similarity) to CysGB, and the N-terminal 264 of which are similar to CbiX. Further evidence that cbiXcysGB is a single gene was provided by SDS-PAGE analysis of the overproduced protein which showed (Fig. 3
) that its molecular mass is consistent with the value (44·3 kDa) calculated from the amino acid sequence. In an experiment similar to that reported previously for the analysis of S. typhimurium cbiK gene (Raux et al., 1997
), plasmids for the expression of the P. freudenreichii cobA and cbiXcysGB genes, either by themselves or together, were constructed and tested for the ability to complement an E. coli cysG deletion mutant (CB302
a). The mutant was complemented by a plasmid (pCR488) bearing both genes but not by a plasmid bearing either cobA (pISA417) or cbiXcysGB (pCR487) by itself. Based on these results, we propose that cbiXcysGB encodes dehydrogenase and/or chelatase activities necessary for cobalamin biosynthesis in P. freudenreichii, although the precise functions of the two domains have not yet been firmly established.
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In S. typhimurium, cbiE, cbiG and cbiH are all individual genes, but in P. freudenreichii their homologues are found in a single ORF (Fig. 1b). Evidence that cbiEGH is a single gene was also provided by SDS-PAGE analysis of the overproduced protein, which showed (Fig. 3
) that its molecular mass is consistent with the value (91·4 kDa) calculated from the amino acid sequence. In Synechocystis sp., cbiG and cbiH are also apparently found in one gene (Kaneko et al., 1996
). CbiH of S. typhimurium has recently been shown to be responsible for C-17 methylation of cobalt-precorrin 3 (Fig. 2a
), leading to formation of the ring-contracted, lactonized intermediate, cobalt-precorrin 4 (Santander et al., 1997
). Although the function of CbiG is unknown, the occurrence of CbiG and CbiH as a single enzyme in two different organisms suggests that CbiG may operate either immediately before or immediately after CbiH. However, the association of CbiE with CbiG and CbiH in P. freudenreichii is not as easily rationalized since it is believed that, based on its similarity to CobL, CbiE catalyses the methylation of C-5 and C-15 but only after methylation of C-11 and C-1, extrusion of acetaldehyde, and reduction of the macrocycle (Fig. 2a
). C-11 methylation is catalysed by CbiF (Roessner et al., 1992
) but the enzyme responsible for C-1 methylation and extrusion of acetaldehyde has yet to be identified. The corresponding process (C-1 methylation and extrusion of acetic acid) in Ps. denitrificans is catalysed by CobF, for which no homologous enzyme exists in S. typhimurium. No gene corresponding to CobF was found within the P. freudenreichii operons reported here. In the anaerobic pathway, these steps are presumably carried out by one of the other methyltransferases, perhaps CbiF, or by CbiD as suggested by Raux et al. (1998)
. On the basis of homology with the corresponding enzymes of Ps. denitrificans, CbiJ is responsible for the NADPH-dependent reduction of the C-18,19 double bond, CbiT catalyses decarboxylation of the C-12 acetate, and CbiC catalyses the final step in the biosynthesis of cobyrinic acid, migration of the methyl group at C-11 to C-12 (Fig. 2a
).
Other B12 biosynthesis genes
In addition to the genes required for the conversion of uroporphyrinogen III to cobyrinic acid, we have isolated a fragment of P. freudenreichii DNA (Fig. 1c) bearing genes encoding enzymes similar to CbiB, CbiP, and CobAadoT of S. typhimurium (Roth et al., 1993
; Suh & Escalante-Semerena, 1993
) involved in the attachment of aminopropanol, amidation of the b, d, e, and g acetate and propionate side chains, and transfer of an adenosyl group to the cobalt metal ion (Fig. 2b
). The CobA adenosyltransferase is also similar to BtuR of E. coli (Lundrigan & Kadner, 1989
), responsible for adenosylation of imported B12. The P. freudenreichii genes for the biosynthesis of adenosylcobalamin from 5-aminolaevulinic acid that have not yet been isolated include the early-pathway genes hemC, hemD and cbiA, and the late-pathway genes cobU, cobS and cobT (see Table 1
for functions). In addition, the gene encoding the enzyme necessary for the reduction of cobalt(II) to cobalt(I) prior to adenosylation has not yet been determined in any organism.
Other P. freudenreichii genes (non-B12)
During our search for B12 biosynthesis genes, we rediscovered partial sequences for most of the P. freudenreichii genes previously isolated, which has allowed the positioning of these genes relative to the sequences described herein. Hashimoto et al. (1996) reported the isolation of an 8 kb fragment of DNA bearing the hemY, hemH, hemB (5-aminolaevulinic acid dehydratase) and hemL (glutamate-1-semialdehyde aminotransferase) genes and two additional, non-B12 biosynthesis genes encoding a probable membrane-associated antibiotic-resistance protein and its regulator. We extended this sequence in the 3' direction and discovered a portion of the gene encoding the 5S subunit of methylmalonyl-CoA transcarboxylase (Thornton et al., 1993a
). Since the genes encoding the 12S and 1·3S subunits of the transcarboxylase are contiguous with the 5S gene (Thornton et al., 1993b
), the structure of the 12 kb fragment of P. freudenreichii DNA bearing all of these genes may be deduced as shown in Fig. 1(d)
.
The 3' end of the fragment bearing most of the B12 biosynthesis genes (Fig. 1b) also harbours the araD and bcp genes encoding proteins similar to L-ribulose-5-phosphate 4-epimerase and bacterioferritin comigratory protein, respectively. Of particular interest, however, is the presence of another operon at the 5' end of this fragment, divergently transcribed from the B12 operon, containing the mutA and mutB genes that encode the two subunits of the adenosylcobalamin-dependent enzyme methylmalonyl-CoA mutase (Marsh et al., 1989
). A similar arrangement of operons occurs in S. typhimurium (Roth et al., 1993
), in which the major B12 operon is divergently transcribed from the operon encoding propanediol dehydratase, encoded by the pduC and pduD genes (Walter et al., 1997
), which is also B12 dependent. In S. typhimurium, B12 is produced only anaerobically (Jeter et al., 1984
) and production of the vitamin is also inhibited by exogenous B12. Both operons are positively regulated by the PocR protein (Bobik et al., 1992
; Rondon & Escalante-Semerena, 1992
), the gene for which lies between the two operons, and whose level is highest under anaerobic conditions in the presence of propanediol. Upstream from the B12 operon lies a B12 box, a short conserved sequence also found before the Ps. denitrificans cobP gene and the E. coli btuB gene (Roth et al., 1993
). B12 has been shown to regulate the synthesis of BtuB by binding to the B12 box in the mRNA, thus interfering with ribosomal binding (Nou & Kadner, 2000
).
By analogy with the regulatory system in S. typhimurium, it is interesting to suggest that the region between the divergently transcribed methylmalonyl-CoA mutase and B12 operons of P. freudenreichii described here (Fig. 1b) would be an ideal location for the observed inhibition of B12 production by exogenous B12 and of the inhibition of propionate and B12 production by oxygen (Quesada-Chanto et al., 1998
; Vorobjeva, 2000
). Methylmalonyl-CoA mutase is a key enzyme in the production of propionic acid (see Vorobjena, 2000, for a review of metabolic pathways in propionibacteria), and a decrease in the enzyme level in the presence of oxygen, by inhibiting synthesis either of the cofactor or of the apoenzyme, would result in decreased propionate levels. In the P. freudenreichii sequence reported here, there is a small ORF (Fig. 1b
, orf1) between the first known gene (cbiL) of the B12 operon and the first gene of the methylmalonyl-CoA mutase operon (mutA). This ORF could encode a protein involved in the regulation of the two operons. The encoded protein, however, has no similarity to PocR or any other known protein. Within orf1, however, there is a 17 base sequence that is a close match to the B12 box sequence (Fig. 4
) which may be responsible for regulation of cobalamin production by exogenous B12 in P. freudenreichii.
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ACKNOWLEDGEMENTS |
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Received 22 October 2001;
revised 28 January 2002;
accepted 27 February 2002.