The Gene Encoding the Biotin Carboxylase Subunit of Escherichia

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol . 261, No. 2, Issue of January 15, p : 855-863,1992 nnted m U.S.A.

The Gene Encoding the Biotin Carboxylase Subunit of Escherichia coli Acetyl-coA Carboxylase*

(Received for publication, April 29, 1991)

Shyr-Jiann Lis and John E. Cronan, Jr.$# From the Departments of $Microbiology and §Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

We report the molecular cloning and DNA sequence of the gene encoding the biotin carboxylase subunit of Escherichia coli acetyl-coA carboxylase. The biotin carboxylase gene encodes a protein of 449 residues that is strikingly similar to amino-terminal segments of two biotin-dependent carboxylase proteins, yeast pyruvate carboxylase and the a-subunit of rat propionyl-CoA carboxylase. The deduced biotin carboxylase sequence contains a consensus ATP binding site and a cysteine-containing sequence preserved in all sequenced bicarbonate-dependent biotin carboxylases that may play a key catalytic role. The gene encoding the biotin carboxyl carrier protein (BCCP) subunit of acetyl-coA carboxylase is located upstream of the biotin carboxylase gene and the two genes are cotranscribed. As previously reported by others, the BCCP sequence encoded a protein of 16,688 molecular mass. However, this value is much smaller than that (22,500 daltons) obtained by analysis of the protein. Amino- terminal amino acid sequencing of the purified BCCP protein confirmed the deduced amino acid sequence indicating that BCCP is a protein of atypical physical properties. Northern and primer extension analyses demonstrate that BCCP and biotin carboxylase are transcribed as a single mRNA species that contains an unusually long untranslated leader preceding the BCCP gene. We have also determined the mutational alteration in a previously isolated acetyl-coA carboxylase ( fabE) mutant and show the lesion maps within the BCCP gene and results in a BCCP species defective in acceptance of biotin. Translational fusions of the carboxyl-terminal 110 or 84 (but not 76) amino acids of BCCP to &galactosidase resulted in biotinated 8- galactosidase molecules and production of one such fusion was shown to result in derepression of the biotin biosynthetic operon.

Acetyl-coA carboxylase catalyses the first committed step in fatty acid synthesis, the synthesis of malonyl-CoA (Alberts and Vagelos, 1972). The overall reaction consists of two distinct half-reactions (Scheme l), the carboxylation of biotin with bicarbonate followed by transfer of the CO, group from carboxy-biotin to acetyl-coA to form malonyl-CoA (Alberts and Vagelos, 1972; Polakis et al., 1974). In Escherichia coli the half-reactions are catalyzed by two different acetyl-coA

*This project was supported by National Institutes of Health Grant AI15650. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

to the GenBankTM/EMBL Data Bank with accession number(s) The nucleotide sequence(s) reported in this paper has been submitted

M80458.

carboxylase subunits, biotin carboxylase and carboxyltransferase (Alberts and Vagelos, 1972; Polakis et al., 1974). A third subunit, biotin carboxyl carrier protein (BCCP),’ carries the essential biotin cofactor covalently bound to a lysine residue proximal to it’s carboxyl terminus (Scheme 1).

Enzymes using biotin as a carboxyl carrier are strongly conserved at the amino acid sequence level (Samols et al., 1988), but the organization of these sequences varies mark- edly. In E. coli acetyl-coA carboxylase and Propionibacterium shermni i transcarboxylase (Samols et al., 1988), the covalently bound biotin, biotin carboxylase, and carboxyltransferase reside in distinct protein subunits, whereas the higher eucaryotic acetyl-coA carboxylases (Takai et al., 1988; Lopez- Casillas et al., 1988) and yeast pyruvate carboxylase (Lim et al., 1988) contain all three components in a single protein chain. Between these extremes lie propionyl-CoA carboxylase which contains two protein subunits (Browner et al., 1989).

In this paper we report the DNA sequence of biotin carboxylase and show that this gene is cotranscribed with the gene encoding the BCCP subunit. Since most recent mechanistic studies of biotin carboxylation have used E. coli biotin carboxylase (Climent and Rubio, 1986; Ogita and Knowles, 1988; Tipton and Cleland, 1988a, 1988b; for review see Knowles, 1989), the sequence is of particular interest. We also report work on the BCCP subunit including the mutational lesion of a previously described acetyl-coA carboxylase mutant (fabE).

EXPERIMENTAL PROCEDURES~

RESULTS

Cloning of the Genes Encoding BCCP and Biotin Carboxyl- ase-During previous work on protein biotination (Cronan, 1989,1990) a synthetic gene segment encoding the carboxyl- terminal 66 amino acid residues of BCCP (Sutton et al., 1977) was assembled (Cronan et al., 1988). This DNA segment was used as a hybridization probe in Southern blots of E. coli chromosomal DNA digested with a variety of restriction enzymes. Strong hybridization was observed to unique DNA fragments under highly stringent conditions indicating a high level of specific base pairing (>85%) between the synthetic DNA and a single chromosomal fragment of each digest (data not shown). A 1.8-kbp PstI-Hind111 fragment was cloned by making a bank of chromosomal fragments in phage M13mplO. M13 was used rather than a plasmid vector since M13 repli-

The abbreviations used are: BCCP, biotin carboxyl carrier protein; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electro- phoresis.

Portions of this paper (including “Experimental Procedures,” Tables 1 and 2, and Figs. 1, 3, and 5) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

855

856 Gene Encoding the Biotin Carboxylase Subunit

ATP *H COS ADP*Pi

BCCP d2-Bccp CARBOXYLTRANSFERASE -

M A L O N Y L CoA ACETYL CoA SCHEME 1

cation produces single-stranded DNA which can be selectively probed in the presence of the double-stranded bacterial chromosomal DNA (Wei and Surzycki, 1986). Three positive clones carrying 1.8-kbp inserts were obtained which appeared identical by restriction mapping (Fig. 1) and partial DNA sequence analysis. DNA sequencing of one of these clones gave a deduced sequence identical to the amino acid sequence reported by Sutton et al. (1977) except that the residue nine amino acids upstream of the biotinated lysine (residue 39 of the Sutton sequence) was Asp rather than Asn (Fig. 2). However since the BCCP species sequenced by Sutton et al. (1977) was known to be a proteolytic fragment of native BCCP, we had cloned only the DNA segment that encodes the carboxyl-terminal half of BCCP (due to occurrence of a Hind111 site within the BCCP gene). The intact gene was obtained by using the cloned fragment to probe the ordered miniset of E. coli chromosomal segments constructed in phage X by Kohara et al. (1987). Only a single clone of the miniset (which covers essentially the entire E. coli chromosome), hybridized. This phage, X 6G3, carries the min 71-72 segment of the E. coli chromosome and the intact BCCP gene was isolated from the phage DNA as a 6-kbp BamHI fragment (Fig. 1). Sequence analysis (Fig. 1) of various subclones of BamHI fragment gave a sequence encoding a 16.7-kDa protein that included the previously reported BCCP carboxyl-terminal sequence (Fig. 2). Following our completion of the sequence two other groups reported the DNA sequence of the BCCP gene. Mizuno (1987) cloned and sequenced several segments of E. coli DNA that attain a conformation in which the helix axis is bent and upon sequence analysis located the BCCP gene downstream from one such sequence (Muramatsu and Mizuno 1989), whereas Alix (1989) cloned and sequenced the BCCP gene of E. coli during his work on two neighboring genes, prmA and panF. Our BCCP sequence is in complete agreement with those reports. The sequence downstream of BCCP encodes biotin carboxylase (see below).

Biotin Carboxylase-The 6-kbp BamHI fragment encoded three proteins as determined by either the maxicell technique or by the phage T7 polymerase expression system (Fig. 3). The apparent molecular masses were 22,50, and 35 kDa. The 22-kDa protein comigrated with [3H]biotin-labeled BCCP and deletions within the BCCP sequence eliminated synthesis of this protein (Fig. 3). Analysis of various deletions (Fig. 3) showed that the 50-kDa protein was encoded by a DNA sequence located immediately downstream of the BCCP gene (Fig. 1). Since biotin carboxylase has a reported molecular weight of 50,000 (Guchhait et al., 1979), we sequenced this downstream DNA segment and obtained a 1350-bp ORF (Fig. 2) encoding a protein of 49,396 Da. Amino-terminal sequencing of homogeneous biotin carboxylase (obtained from Dr. V. Rubio (University of Valencia, Spain); Climent and Rubio, 1986) gave a 31-amino acid sequence that exactly matched the deduced sequence (Table 1). Moreover, multicopy plasmids carrying this DNA segment overproduced carboxylase activity 2-3-fold when transcription was from the native

Gene Encoding the Biotin Carboxylase Subunit 857

the known modest expression level of BCCP (-1000 copies/ cell) (Cronan, 1989) and the purification factor required to obtain homogenous biotin carboxylase (Guchhait et al., 1974).

Transcription of the BCCP and Biotin Carboxylase Genes- The very short (10 bp) intragenic region between the genes encoding the two proteins (Fig. 2) suggested that the BCCP and biotin carboxylase genes are cotranscribed. Cotranscrip- tion was tested by insertion of a DNA segment containing strong transcriptional terminators into the BCCP gene of the 6 kbp BamHI fragment carried by pLS1. This insertion abol- ished the synthesis of both BCCP and biotin carboxylase in the maxicell system indicating that the two genes share the same promoter (Fig. 3B).

An mRNA species of -2.2 kb was detected by Northern blot analyses using probes specific for BCCP or biotin carboxylase or a probe containing sequences of both genes (Fig. 4A). Essentially identical results were obtained using three different RNA extraction methods.

The transcriptional initiation site for the 2.2-kb transcript was located by primer extension. The first primer was complementary to the mRNA sequence 42 bp upstream of the BCCP initiator methionine and gave a 217-nucleotide primer- extended product indicating an 5"untranslated leader mRNA sequence of 319 nucleotides (data not shown). This result was confirmed by extension of a second primer beginning 238 bp upstream of the BCCP coding sequence (Fig. 4B). Thus, this mRNA has an unusually long leader sequence (Fig. 2) and includes a transcript of a 98-bp DNA sequence reported to have an intrinsically bent structure (Mizuno, 1987; Mura- matsu and Mizuno, 1989). Moreover, the transcript includes a short ORF of 188 bp that overlaps the first eight amino acids of BCCP. The length of the BCCP-biotin carboxylase mRNA indicates that transcription termination occurs between the carboxylase gene and the downstream panF gene. A sequence that may be a factor-independent transcription terminator is found just upstream of the proposed panF promoter (Jackowski and Alix, 1990).

Correspondence of the Derived BCCP Sequence with the Authentic Protein-In their reports of the deduced amino acid sequence of the BCCP gene Muramatsu and Mizuno (1989) and Alix (1989) failed to note a large discrepancy between the deduced and measured molecular masses of BCCP. The DNA sequence predicts a protein of 16,688 Da, whereas the monomeric protein has been reported to have a molecular mass of 22,500 Da by SDS-PAGE (Fall and Vagelos, 1972; Murtif et al., 1985; Cronan, 1990) and 21,800 Da by gel filtration in guanidine HCl (Fall and Vagelos, 1972). Moreover, the native protein was reported to be a dimer of molecular mass 45 kDa (Fall and Vagelos, 1972).

The large difference between the measured and deduced molecular weights of BCCP suggested that the deduced amino acid sequence could be incorrect. Specifically, several putative initiation codons in different reading frames are found upstream of the initiation codon and thus a sequencing error giving a frame-shift seemed possible. To resolve this point we purified BCCP from wild type E. coli K-12 and determined the amino-terminal amino acid sequence of the band migrat- ing at 22.5 kDa on SDS-PAGE. The sequence obtained by automated Edman sequencing (Table 1) exactly matched the deduced sequence over 23 residues. The amino acid composition previously determined for BCCP from E. coli strain B (Fall and Vagelos, 1972) closely matched the composition predicted from the DNA sequence (Table 2) and the predicted and determined isoelectric points are identical (pH 4.5). More- over, when the amino acid composition of the segment of BCCP determined by direct amino acid sequencing (i.e. the

A.

-2.2Kb

- - FIG. 4. Northern blot and primer extension analyses. A,

Northern blot analysis. Lanes I and 2, total RNA preparations from strain BMH71-18 (lacking plasmids, lane I ) or strain BMH71-18 carrying pLSl which contains the entire 6-kbp BamHI chromosomal inset (Fig. 2). The probe was the 1.8-kbp HindIII-PstI fragment (Fig. 2) which carries both BCCP and biotin carboxylase sequences. Lanes 3 and 4, a total RNA preparation from BMH71-18 (lacking plasmids) was run in two adjacent lanes of a gel and then transferred to a hybridization membrane. The membrane was cut in half (each half containing one lane). The membrane half derived from lane 3 was probed with a 483-bp NsiI-KpnI fragment specific for BCCP, whereas the membrane of lane 4 was probed with a 768-bp SstII-EcoRV probe specific for biotin carboxylase (Fig. 2). Lanes 5 and 6, RNA preparations from strain BMH71-18 carrying pLSlOl which contains the right hand NsiI-BamHI fragment (Fig. 2) with transcription from the vector lac promoter. Lane 5, plus IPTG induction; lane 6, no induction. Lanes 7 and 8 are a repeat of lanes 2 and 1 , respectively. The BCCP-specific probe (see above) was used. The hybridizing bands a t the top of the gel are due to plasmid DNA which accompanies the RNA in the procedure of Kornblum et al. (1988) and shows variable transfer to the membrane depending on the transfer protocol. In each gel RNA samples derived from equivalent amounts of cells were

bp 5' to the initiation codon of BCCP was 32P-labeled at the 5' end loaded. B, a 30-mer oligonucleotide complementary to a region 238

and annealed to 40 pg of total RNA (see "Experimental Procedures"). Hybrids were extended by AMV reverse transcriptase. Lane I , primer extension using RNA prepared from strain BMH71-18 carrying plasmid pLS1. Lane 2, RNA prepared from the same strain lacking plasmid. Lanes A, T, C, G are from a sequencing reaction run with the same primer and loaded on the same gel. The letters denote the dideoxynucleotide triphosphate added to the sequencing reaction of that lane (e.g. A denotes ddATP).

data of Sutton et al. (1977) plus our amino-terminal sequence) is subtracted from the composition of the intact protein, the resulting composition closely matches that obtained for residues 24-74 of the deduced sequence (Table 2). A second line of evidence stems from construction of a DNA segment in which the 468-bp sequence encoding the 16.7-kDa protein was the only ORF. A plasmid containing the 584-bp NsiI-SstII


segment downstream of a T7 promoter was constructed such that the only initiation codon (ATG or GTG) between the promoter and the BCCP amino terminus was that identified by amino-terminal sequencing (Table 1). This plasmid encoded the putative 22.5-kDa species (Fig. 3C). Since the SstII site is located within the biotin carboxylase gene (at codons 9-11) and lies only 41 bp downstream of the BCCP terminator codon, the putative 22.5-kDa protein must be encoded by an ORF of only 468 bp. Moreover, all reading frames of the sequence upstream of the start site contain many codons rarely used in E. coli indicating the DNA segment does not encode a protein (Gribskov et al., 1984). These data taken together indicate that BCCP is a 16.7-kDa protein of unusual physical properties.

Molecular Characterization of the fabE Mutation-Harder et al. (1972) isolated mutants of E. coli conditionally defective in total fatty acid synthesis. One of these strains was subse- quently shown to be defective in acetyl-coA carboxylase activity as assayed in uitro (Silbert et al., 1976). The growth and enzymatic phenotypes were conditional on temperature; being normal at 30 "C but defective at higher temperatures (37 and 42 "C). The characterization of this mutant was inconclusive; the mutant acetyl-coA carboxylase seemed defective in both BCCP biotin content and biotin carboxylase activity as assayed on free biotin (Silbert et al., 1976). More- over, since all analyses was done in the original heavily mutagenized background, the possibility of multiple muta- tions existed.

We isolated DNA carrying the fubE mutational lesion by polymerase chain reaction amplification of strain L8 chromosomal DNA. Upon sequence analyses a single mutational change was found, a G to A transversion that converted GlylW of BCCP to Ser. Since Glylo0 is close to the site of biotin attachment and a glycine residue is found at the analogous positions in all biotinated proteins (Samols et al., 1988), it seemed that the phenotype of the mutant might be due to deficient biotination of BCCP.

Indeed, labeling of strain L8 showed that the strain incor- porated less [3H]biotin into BCCP than a wild type strain, and the biotination defect was more severe at higher growth temperatures (Fig. 5 A ) . To examine this point more directly we examined strains encoding protein fusions between 0- galactosidase and either the fabE or wild type biotination domains.

Protein Biotination of Protein Fusions-We previously showed that in uitro constructed translational fusion proteins are biotinated efficiently by E. coli (Cronan, 1990; Reed and Cronan, 1991). These bipartite fusion proteins consist of an amino-terminal heterologous reporter protein (e.g. P-galactosidase) fused to a carboxyl-terminal biotination sequence ("biotin tail"). Most prior work had been done with the 1.3 S biotin protein from Propionibacterium shermanii transcarboxylase (Cronan, 1990) and thus we first examined fusions to wild type BCCP sequences.

Genes encoding various segments of BCCP fused to an enzymatically active @-galactosidase were constructed. We found that fusion proteins containing the last 84, 87, or 110 amino acids of BCCP were efficiently biotinated, whereas a fusion with the 76 BCCP carboxyl-terminal residues failed to be biotinated (Fig. 5B) .

0-Galactosidase fusions to the last 110 residues of the fabE mutant BCCP protein were found to be poorly biotinated (Fig. 5B) particularily in cells grown at 42 "C and thus the Gly"" to Ser mutation resulted in a temperature-sensitive defect in biotination. The growth phenotype of the fabE mutant could not be complemented by introduction of a

plasmid overproducing biotin ligase (data not shown). Regulation of Biotin Operon Expression-A fusion of the

carboxyl-terminal 110 residues of the wild type BCCP gene to 6 amino acid residues of 0-galactosidase was used to examine the effect of BCCP production on transcription of the biotin synthetic operon. This previously had been studied with an heterologous biotinated protein, the P . shermanii 1.3 S protein (Cronan, 1988,1989). As expected from these results and the model of biotin operon regulation (Cronan, 1989) high level expression of the BCCP fusion protein gave a 40- fold derepression of biotin operon expression (data not shown).

DISCUSSION

We have characterized the structure and expression of two cotranscribed genes that encode subunits of E. coli acetyl- CoA carboxylase, BCCP and biotin carboxylase. BCCP and biotin carboxylase function as a complex (Alberts and Vage- los, 1972; Guchhait et al., 1979) presumably of defined stoi- chiometry and thus cotranscription could facilitate efficient formation of the complex. The genes encoding the two subunits of the carboxyltransferase component of acetyl-coA carboxylase (Guchhait et al., 1979) are not closely linked to the BCCP and biotin carboxylase genes (data not shown).

The deduced 499-amino acid sequence shows striking similarity to the amino-terminal regions of two proteins with biotin carboxylase activity, yeast pyruvate carboxylase (Lim et al., 1988), and the a-subunit of rat propionyl-CoA carboxylase (Browner et al., 1989) (Fig. 6). Essentially the entire E. coli sequence could be aligned with both proteins. The a- subunit of propionyl-CoA carboxylase showed 49% identical residues with the E. coli protein, whereas the value for PYN- vate carboxylase was 45% (the two eucaryotic proteins are 42% identical). The three sequences are interrelated such that

KYLE RFLD KFID

5~LQIRl~N::~"NlHYLE~~LGLQEK IPFLLTLLTNP YWGTIIDDTPOLPOM-..

IPLLREVIINTR K DISTKFLSDVYPDGFK...

FIG. 6. Alignment of the entire E. coli biotin carboxylase (BTN) sequence with the amino-terminal sequences of yeast pyruvate carboxylase (PYR) beginning at residue 18 and the a-subunit of rat propionyl-CoA carboxylase (PRC) beginning at residue 55 (the first 43 residues comprise the leader peptide cleaved upon mitochondrial entry). Residues identical in all three sequences are boxed. The 2 cysteine residues (C230 and C336 of biotin carboxylase) discussed in the text are marked (*).


when a residue present in biotin carboxylase is not conserved in propionyl-CoA carboxylase, it is often conserved in pyruvate carboxylase and vice versa (Fig. 6). By the criteria of Doolittle (1981), these proteins are clearly related by common ancestry. As discussed below, a region of close similarity between the E. coli protein and the acetyl-coA carboxylases of rat and chicken was also observed.

The alignments of Fig. 6 allow functional assignment of the sequences of the eucaryotic biotin-dependent carboxylase. Such analysis had progressed furthest with yeast pyruvate carboxylase where Lim et al. (1988) inferred putative ATP and bicarbonate binding sites to the amino-terminal half of the molecule by similarity to carbamyl-phosphate synthetase, an enzyme utilizing ATP and bicarbonate (but not biotin), and to other biotin-dependent carboxylases. The extensive regions of identical sequence in biotin carboxylase and yeast pyruvate carboxylase (Fig. 6) strengthens the interpretation of Lim et al. (1989) that the amino-terminal half of pyruvate carboxylase catalyses the biotin carboxylation partial reaction. We also observed strong similarity between biotin carboxylase and the a-subunit of rat propionyl-CoA carboxylase (Fig. 6). The functional form of the latter enzyme is a complex of a- and @-subunits. By analogy to a bacterial propionyl-CoA carboxylase (Haase et al., 1982) the biotin carboxylase partial reaction was thought to reside in the a-subunit together with the biotinated domain, whereas the @-subunit (Kraus et al., 1986) catalyzed the carboxyltransferase reaction (Samols et al., 1988). Our sequence alignment provides strong support for this interpretation since the a-subunit seems composed of only biotin carboxylase and BCCP-like domains. Assignment of the carboxyltransferase activity to the @-subunit is supported by the previously reported (Samols et al., 1988) similarity between this protein and the 12 S carboxyltransferase subunit of transcarboxylase (note that the ,&subunit is often mistakenly referred to as the a-subunit by Samols et al.).

The assignment of the ATP and (perhaps) bicarbonate binding regions of biotin carboxylase seems straightforward based on previous interpretations of the similarity of yeast pyruvate carboxylase to carbamyl-phosphate synthetase (Lim et al., 1988). Biotin carboxylase likewise shows strong similarity to E. coli carbamyl-phosphate synthetase (Fig. 7). The glycine-rich region of biotin carboxylase centered at residue 167 is very similar to the adenylate kinase ATP binding site (Reinstein et al., 1988) and to both of the ATP binding sites of E. coli carbamyl-phosphate synthetase (Post et al., 1989). Moreover, site-directed mutagenesis of the glycine-rich re-

FIG. 7. Identities between biotin carboxylase (BTNCARB, residues 160-348), rat acetyl-coA carboxylase (ACC-RAT, residues .301- 496) (Lopez-Casillas et al., 1988). and E. coli carbamyl-phosphate synthetase (CARPSYN, residues 706-900) (Nyunoya and Lusty, 1983). Identical residues are bored. C230 and C336 of biotin carboxylase are marked (*). Note that a very similar alignment could be obtained within the amino-terminal half of E. coli carbamyl- phosphate synthetase due to the dupli- cated nature of this protein. Biotin carboxylase shows 22-23% identity with each of the two halves of the synthase protein. The sequence of the chicken acetyl-coA carboxylase (Takai et al., 1988) is identical to the rat enzyme in this region.

gions of both proteins is known to alter ATP binding (Rein- stein et al., 1988; Post et al., 1989).

Downstream of the putative ATP site lies a region highly conserved in ATP-dependent biotin carboxylases, but not found in carbamyl-phosphate synthetases (Fig. 7). Tipton and Cleland (1988a, 1988b) have reported evidence for involve- ment of a proton donor/acceptor group in the transfer of C02 from the postulated carboxyphosphate intermediate to biotin. The chemical behavior of this group is characteristic of a thiol, and thus we have examined the carboxylase sequences for a conserved cysteine residue. The three enzyme sequences compared in Fig. 6 contain 2 conserved cysteine residues at coordinates 232 and 343 (residues C230 and C336 of biotin carboxylase). Given substitution of aliphatic hydrophobic amino acids, sequences very similar to that around C230 are also found in the chicken and rat acetyl-coA carboxylases (Fig. 7, centered at coordinate 85). Moreover, the spacings of these sequences relative to the putative carboxylase ATP binding sites are also very similar to that found in biotin carboxylase. In contrast, the C336 region is not conserved in the eucaryotic acetyl-coA carboxylases (Fig. 7) indicating these residues do not play a key role in biotin carboxylation. The case for a specific role for the C230 region in COn fixation from bicarbonate is strengthened by the finding that these sequences are not conserved in two enzymes which obtain the COz group of carboxy-biotin from a dicarboxylic acid (rather than bicarbonate), oxalacetate decarboxylase (Schwarz et al., 1988), and transcarboxylase (Samols et al., 1988). The location of the C230 region close to the putative ATP and bicarbonate binding regions suggests a linear array of amino acid residues important in catalysis and that the C230 region may also include sequences involved in biotin binding.

Although BCCP is a protein of 16.7 kDa, it behaves as a protein of much larger molecular mass (-22 kDa) on SDS- PAGE. This anomaly seems almost certainly due to the unusual amino acid sequence between residues 34 and 101 where almost half of the residues are proline or alanine. Very similar Pro/Ala-rich sequences are known to be responsible for discrepancies between the actual (66 kDa) and SDS- PAGE-derived (78-83 kDa) molecular masses of E. coli dihy- drolipoamide acetyltransferase (AceF protein) (Miles et al., 1988) and several other lipoated proteins. In the case of the AceF protein, it is known that progressive deletion of the Pro/Ala sequences from a modified gene encoding this enzyme results in convergence of the actual and SDS-PAGE molecular weight values. For a modified AceF protein lacking the Pro/

1 8 0 V IKQEEV RIKDIR EQGVTK

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Ala sequence the actual and SDS-PAGE values agreed within 3%, whereas a protein containing a %-residue Pro/Ala-rich sequence gave an SDS-PAGE value 14% greater than the actual molecular weight (Miles et al., 1988). The discrepancy seen with BCCP is larger (-35%) than that seen with AceF. This may be attributed to the fact that Pro/Ala-rich sequences comprise about 20% of the BCCP sequence, but only about 10% of the AceF sequence. Pro/Ala sequences are known to be extraordinary mobile (Radford et al., 1989), and the large molecular radius resulting from this mobility (Easom et al., 1989) probably explains the molecular weights of 22,000 and 45,000 for the monomeric and dimeric forms of BCCP obtained by gel filtration (Fall and Vagelos, 1972). Proteolytic cleavage within the mobile Pro/Ala sequences during purification explains the diversity of small forms of BCCP that confused early work on this protein (for review see Alberts and Vagelos, 1972). The proteolytic product sequenced by Sutton et al. (1977) resulted from cleavage between alanine residues 74 and 75, whereas the amino acid composition of a higher molecular weight (10, 400) species of BCCP (Nervi et al., 1971) is consistent with cleavage between Ala residues 61 and 62. The other species reported (Fall and Vagelos, 1973) can be attributed to cleavages within the Pro/Ala sequences located close to the amino terminus. These carboxyl-terminal peptides interact with the biotin carboxylase and carboxyltransferase subunits, but much more weakly than does the intact BCCP protein (Fall and Vagelos, 1973). Thus, a function of the BCCP amino terminus is to tether BCCP to the other acetyl-coA carboxylase subunits, whereas the central Pro/Ala-rich sequence allows the carboxyl-terminal biotinated domain to move between the subunit active sites (Lynen, 1972).

Acknowledgments-We thank Dr. V. Rubio for his generous gift of biotin carboxylase and Dr. J.-H. Alix for a preprint of his manuscript.

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126,1351-1354

8 7 , 1 1 4 - 1 2 6

A. 84,4767-4771

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Gotoh, T. (1987) Gene (Amst.) 61,63-74

4325

4331


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862 Gene Encoding the Biotin Carboxylase Subunit NsiI \ Hind111

'N CAR6

A 1 2 3 . 1

- GL3

BC - I

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Gene Encoding the Biotin Carboxylase Subunit

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29

B. 1 2 3 4 5 6 7

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863

Documents

The Gene Encoding the Biotin Carboxylase Subunit of Escherichia