Interchangeable Enzyme Modules

Biotin carboxyl carrier protein (BCCP) is the small biotinylated subunit of Escherichia coli acetyl-CoA carboxylase, the enzyme that catalyzes the first committed step of fatty acid synthesis. E. coli BCCP is a member of a large family of protein domains modified by covalent attachment of biotin. In most biotinylated proteins, the biotin moiety is attached to a lysine residue located about 35 residues from the carboxyl terminus of the protein, which lies in the center of a strongly conserved sequence that forms a tightly folded anti-parallel β-barrel structure. Located upstream of the conserved biotinoyl domain sequence are proline/alanine-rich sequences of varying lengths, which have been proposed to act as flexible linkers. In E. coli BCCP, this putative linker extends for about 42 residues with over half of the residues being proline or alanine. I report that deletion of the 30 linker residues located adjacent to the biotinoyl domain resulted in a BCCP species that was defective in function in vivo, although it was efficiently biotinylated. Expression of this BCCP species failed to restore normal growth and fatty acid synthesis to a temperature-sensitiveE. coli strain that lacks BCCP when grown at nonpermissive temperatures. In contrast, replacement of the deleted BCCP linker with a linker derived from E. coli pyruvate dehydrogenase gave a chimeric BCCP species that had normal in vivo function. Expression of BCCPs having deletions of various segments of the linker region of the chimeric protein showed that some deletions of up to 24 residues had significant or full biological activity, whereas others had very weak or no activity. The inactive deletion proteins all lacked an APAAAAA sequence located adjacent to the tightly folded biotinyl domain, whereas deletions that removed only upstream linker sequences remained active. Deletions within the linker of the wild type BCCP protein also showed that the residues adjacent to the tightly folded domain play an essential role in protein function, although in this case some proteins with deletions within this region retained activity. Retention of activity was due to fusion of the domain to upstream sequences. These data provide new evidence for the functional and structural similarities of biotinylated and lipoylated proteins and strongly support a common evolutionary origin of these enzyme subunits. Biotin carboxyl carrier protein (BCCP) is the small biotinylated subunit of Escherichia coli acetyl-CoA carboxylase, the enzyme that catalyzes the first committed step of fatty acid synthesis. E. coli BCCP is a member of a large family of protein domains modified by covalent attachment of biotin. In most biotinylated proteins, the biotin moiety is attached to a lysine residue located about 35 residues from the carboxyl terminus of the protein, which lies in the center of a strongly conserved sequence that forms a tightly folded anti-parallel β-barrel structure. Located upstream of the conserved biotinoyl domain sequence are proline/alanine-rich sequences of varying lengths, which have been proposed to act as flexible linkers. In E. coli BCCP, this putative linker extends for about 42 residues with over half of the residues being proline or alanine. I report that deletion of the 30 linker residues located adjacent to the biotinoyl domain resulted in a BCCP species that was defective in function in vivo, although it was efficiently biotinylated. Expression of this BCCP species failed to restore normal growth and fatty acid synthesis to a temperature-sensitiveE. coli strain that lacks BCCP when grown at nonpermissive temperatures. In contrast, replacement of the deleted BCCP linker with a linker derived from E. coli pyruvate dehydrogenase gave a chimeric BCCP species that had normal in vivo function. Expression of BCCPs having deletions of various segments of the linker region of the chimeric protein showed that some deletions of up to 24 residues had significant or full biological activity, whereas others had very weak or no activity. The inactive deletion proteins all lacked an APAAAAA sequence located adjacent to the tightly folded biotinyl domain, whereas deletions that removed only upstream linker sequences remained active. Deletions within the linker of the wild type BCCP protein also showed that the residues adjacent to the tightly folded domain play an essential role in protein function, although in this case some proteins with deletions within this region retained activity. Retention of activity was due to fusion of the domain to upstream sequences. These data provide new evidence for the functional and structural similarities of biotinylated and lipoylated proteins and strongly support a common evolutionary origin of these enzyme subunits. acetyl-CoA carboxylase biotin carboxyl carrier protein isopropyl-β-D-thiogalactopyranoside 5-bromo-4-chloro-3-indoyl-β-d-galactoside pyruvate dehydrogenase Biotin and lipoic acid are vitamins that play essential roles in central metabolism. The biological activities of both coenzymes are dependent upon their covalent attachment to their cognate proteins (1Perham R.N. Annu. Rev. Biochem. 2000; 69: 961-1004Crossref PubMed Scopus (482) Google Scholar). In both cases, the site of coenzyme attachment is the ε-amino group of a lysine residue centrally located within conserved sequences of ∼70 amino acid residues that fold to form discrete protein domains. The three-dimensional structures of both biotinoyl (2Athappilly F.K. Hendrickson W.A. Structure. 1995; 3: 1407-1419Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 3Reddy D.V. Shenoy B.C. Carey P.R. Sonnichsen F.D. Biochemistry. 2000; 39: 2509-2516Crossref PubMed Scopus (48) Google Scholar, 4Roberts E.L. Shu N. Howard M.J. Broadhurst R.W. Chapman-Smith A. Wallace J.C. Morris T. Cronan J.E., Jr. Perham R.N. Biochemistry. 1999; 38: 5045-5053Crossref PubMed Scopus (69) Google Scholar, 5Yao X. Wei D. Soden C., Jr. Summers M.F. Beckett D. Biochemistry. 1997; 36: 15089-15100Crossref PubMed Scopus (67) Google Scholar, 6Yao X. Soden C., Jr. Summers M.F. Beckett D. Protein Sci. 1999; 8: 307-317Crossref PubMed Scopus (26) Google Scholar) and lipoyl domains (7Dardel F. Davis A.L. Laue E.D. Perham R.N. J. Mol. Biol. 1993; 229: 1037-1048Crossref PubMed Scopus (120) Google Scholar, 8Green J.D. Laue E.D. Perham R.N. Ali S.T. Guest J.R. J. Mol. Biol. 1995; 248: 328-343PubMed Google Scholar, 9Ricaud P.M. Howard M.J. Roberts E.L. Broadhurst R.W. Perham R.N. J. Mol. Biol. 1996; 264: 179-190Crossref PubMed Scopus (60) Google Scholar) from several biological sources have been determined, and the structures are largely superimposable. Indeed, Reche and Perham (10Reche P. Perham R.N. EMBO J. 1999; 18: 2673-2682Crossref PubMed Scopus (67) Google Scholar) have succeeded in altering the Escherichia coliacetyl-CoA carboxylase (ACC)1biotinoyl domain such that the normally absolutely specific lipoate protein ligase will attach lipoic acid to various mutant biotinoyl domains. Therefore, it that enzyme that very a common structural domain to the essential and and play essential roles in fatty acid synthesis and amino acid whereas lipoylated and and are for function of the acid and (1Perham R.N. Annu. Rev. Biochem. 2000; 69: 961-1004Crossref PubMed Scopus (482) Google Scholar). the biotinoyl and lipoyl domains are to act as that of a (1Perham R.N. Annu. Rev. Biochem. 2000; 69: 961-1004Crossref PubMed Scopus (482) Google Scholar). a small is by attachment of the carboxyl of the coenzyme to the ε-amino group of a lysine residue located at the of a first proposed Protein Sci. PubMed Scopus Google Scholar) this the of the coenzymes a significant a is by the proline sequences adjacent to the lipoyl domains that act as flexible (1Perham R.N. Annu. Rev. Biochem. 2000; 69: 961-1004Crossref PubMed Scopus (482) Google Scholar). In lipoyl having only a domain, the domain forms the amino terminus of the protein, and the linker the domain to the domain of the In proteins having lipoyl such as E. coli pyruvate dehydrogenase which lipoyl the domains are in at the amino terminus with the domains from and from the domain proteins are the of the lipoyl domain the modified domain is located at the carboxyl and a proline sequence is upstream of the domain carboxylase catalyzes the first step in fatty acid the synthesis of from acetyl-CoA P.R. The Scholar) is a and all biotin the attached to for enzyme activity. In E. coli and the functional enzyme of of of P. E. J. J. Biol. Full Text PDF PubMed Google Scholar, Cronan J.E., Jr. J. Biol. Full Text PDF PubMed Google Scholar, Cronan J.E., Jr. J. Biol. Full Text PDF PubMed Google Scholar). The biotin is attached to the biotin carboxyl carrier protein (BCCP) the biotinylated protein of this In the first the of the attached biotin moiety is by the biotin carboxylase The carboxyl group is from biotin to acetyl-CoA to by a of proteins and to as are in the of J. X. T. Biochem. 2000; PubMed Google Scholar). The of the biotinoyl domains of the E. coli acetyl-CoA BCCP (2Athappilly F.K. Hendrickson W.A. Structure. 1995; 3: 1407-1419Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 4Roberts E.L. Shu N. Howard M.J. Broadhurst R.W. Chapman-Smith A. Wallace J.C. Morris T. Cronan J.E., Jr. Perham R.N. Biochemistry. 1999; 38: 5045-5053Crossref PubMed Scopus (69) Google Scholar, 5Yao X. Wei D. Soden C., Jr. Summers M.F. Beckett D. Biochemistry. 1997; 36: 15089-15100Crossref PubMed Scopus (67) Google Scholar, 6Yao X. Soden C., Jr. Summers M.F. Beckett D. Protein Sci. 1999; 8: 307-317Crossref PubMed Scopus (26) Google Scholar) and a protein, the subunit D.V. Shenoy B.C. Carey P.R. Sonnichsen F.D. Biochemistry. 2000; 39: 2509-2516Crossref PubMed Scopus (48) Google have been In both cases, the structures of the protein segments located upstream of the biotinoyl domain due to their of structures in these protein segments by or the similarities the biotinoyl and lipoyl it that the putative biotinoyl domain linker play roles to of the lipoyl domain linkers. an this is that the sequences of biotinoyl and lipoyl for a residues in the of some biotinoyl proteins, small residues or have and the of lipoyl proteins are in residues are the biotinoyl linkers. the the conserved domain it that the conserved proline/alanine-rich of the amino acid sequences The E. coli have been and the data that the of proline and in an (1Perham R.N. Annu. Rev. Biochem. 2000; 69: 961-1004Crossref PubMed Scopus (482) Google Scholar, J.D. Perham R.N. E. J. Biol. Full Text PDF PubMed Google Scholar, Guest J.R. Perham R.N. PubMed Scopus Google Scholar, Guest J.R. Perham R.N. J. Mol. Biol. PubMed Scopus Google Scholar, R.N. Roberts PubMed Scopus Google Scholar, Laue E.D. Perham R.N. Guest J.R. Biochem. J. PubMed Scopus Google Scholar, Perham R.N. E. PubMed Scopus Google Scholar, Laue E.D. Perham R.N. E. J. Biol. 264: Full Text PDF PubMed Google Scholar, Laue E.D. Perham R.N. Guest J.R. Biochemistry. PubMed Scopus Google Scholar, Guest J.R. Protein 1993; PubMed Scopus Google Scholar). In the E. coli the linker are to and to The of these linker was first by of the R.N. Roberts PubMed Scopus Google Scholar, Laue E.D. Perham R.N. Guest J.R. Biochem. J. PubMed Scopus Google Scholar). the very of the E. coli a of are that with of the lipoyl domains within the the in the are the as of linker J.D. Perham R.N. E. J. Biol. Full Text PDF PubMed Google Scholar, Laue E.D. Perham R.N. E. J. Biol. 264: Full Text PDF PubMed Google Scholar). In this I report that a large deletion of the putative linker region of the E. coli BCCP subunit in a protein of activity in biological activity was upon of a sequence derived from the first linker of the E. coli that the lipoyl and biotinoyl linker have and of this chimeric protein and of the BCCP showed that only a of the linker was for biological activity and that sequences to provide linker function. The linker was in due to a the of the linker was by the and and this to PubMed Scopus Google Scholar) with the and was such that upon a the of the linker was by and and was to PubMed Scopus Google Scholar) with to the resulted in of a The the and of the with or the in and with the J.E., Jr. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) with and in a The and to a sequence the due to a at the of the was removed by the with and a of and to the linker also derived by the sequence was by and and this was with and a new site and retained the site at of the a to the sequence adjacent to the biotinoyl domain of the wild type was by and and this to with and a site and the in this in a new of deletions within the region the chimeric linker by by at to In most cases, or both of the from in or by with the or by with in to the The to activity of the was to whereas removed by the and the enzyme to the will to these are The as and and and and and and and and and The resulted from the as a deletion in of of the wild type BCCP linker region also The the BCCP resulted from of with by The was and the was with with and The the BCCP resulted from the and and the to with was such that The and BCCP by of the site of the The of PubMed Google Scholar) was to the by for to both and The was by and The of a that the was by of the of the site of PubMed Scopus Google Scholar). The was by by to the of the the and BCCPs a by the and by with and The was to and with showed of of the in the and and in a to The was with and to The and derived from that by of the of PubMed Google Scholar) the site of the The was removed by with to to The resulted from of the as that the removed by and the was to and with The for with the in the that of The the was by the and and the to with The the chimeric protein was from J.E., Jr. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) and D. J. Biol. Full Text PDF PubMed Google Scholar) by of with The was with and and to of was with and within the and a by the and was by The of most of the strain which a fusion and from an was by of strain J. PubMed Google Scholar) with with for to and to the The and and of this with and the The at that very the the in whereas that both of is The was to from the which is for BCCP in and was to BCCP by from the BCCP was of this protein is coli and also to by of by by the of the of with was in the enzyme with the at of was and the was at for by to the with was in the enzyme with of by at 30 for and to inactive the of the was with the the J.E., Jr. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) as of protein by with and as of with the BCCP was by of strain a wild type strain and of Escherichia of Scholar) in was by of the of Cronan J.E., Jr. J. Biol. Full Text PDF PubMed Google Scholar) E. J. PubMed Scopus Google Scholar) with the The structures of the BCCP biotin domain at residues and although these residues and the residues to are the residues of the tightly folded domain (2Athappilly F.K. Hendrickson W.A. Structure. 1995; 3: 1407-1419Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 4Roberts E.L. Shu N. Howard M.J. Broadhurst R.W. Chapman-Smith A. Wallace J.C. Morris T. Cronan J.E., Jr. Perham R.N. Biochemistry. 1999; 38: 5045-5053Crossref PubMed Scopus (69) Google Scholar, 5Yao X. Wei D. Soden C., Jr. Summers M.F. Beckett D. Biochemistry. 1997; 36: 15089-15100Crossref PubMed Scopus (67) Google Scholar, 6Yao X. Soden C., Jr. Summers M.F. Beckett D. Protein Sci. 1999; 8: 307-317Crossref PubMed Scopus (26) Google Scholar). In the was modified to a site that and The this new site and the site was deleted by by with the and These removed most of the putative linker that this to an BCCP the to the such in and have a significant such that N. J. PubMed Scopus Google Scholar). by of the biotin A. Cronan J.E., Jr. Biochem. Sci. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, A. Morris Wallace J.C. Cronan J.E., Jr. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, J.E., Jr. J. Biol. Full Text PDF PubMed Google Scholar). of a biotin protein in of the biotin due to of by protein J.E., Jr. J. Biol. Full Text PDF PubMed Google Scholar). in is by of a strain a fusion in which a biotin of E. J. PubMed Google Scholar). The biotin is by of a In the and the of a biotin domain in whereas the that biotin in biotin was in the of the deletions and all by to have sequences to In the case of the showed an that removed 30 residues of the putative BCCP linker and resulted from of the The function of the and the in this in vivo as J.E., Jr. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). In the an that the BCCP at a with that from the wild type The E. coli mutant which a BCCP that is upon to In the of of a functional BCCP strain to whereas of a functional protein growth of the growth was to about of the normal of BCCP J.E., Jr. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). the wild type at and at 42 the BCCP was in strain the strain very at and failed to at 42 and from J.E., Jr. J. Biol. Full Text Full Text PDF PubMed Scopus Google the growth of the strain at of with Therefore, deletion of the BCCP linker residues resulted in a protein that was activity in to the role of the linker region in BCCP function. The first was a deletion this was due to a of in the sequence the was to a linker sequence for that of BCCP and a was and the sequence of the linker was to In this residues of the putative linker of BCCP with the sequence that the lipoyl domains of E. coli pyruvate dehydrogenase linker was over the coli was that of the proline/alanine-rich region of BCCP, and no or was to this linker The to of the amino acid of the new linker biological function of the chimeric BCCP, of deletions that resulted in of function the of the the the chimeric BCCP was with segments of the BCCP sequence in to restore function. The was that the chimeric BCCP was functional at both and 42 in vivo, that the proline/alanine-rich of BCCP and linker in a this and the and of the both in and as it that upon structural the proline/alanine-rich region of BCCP to a of deletions within and upstream of the linker region of the chimeric protein by of the These deletions all upstream of the first residue of the conserved domain (2Athappilly F.K. Hendrickson W.A. Structure. 1995; 3: 1407-1419Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 4Roberts E.L. Shu N. Howard M.J. Broadhurst R.W. Chapman-Smith A. Wallace J.C. Morris T. Cronan J.E., Jr. Perham R.N. Biochemistry. 1999; 38: 5045-5053Crossref PubMed Scopus (69) Google Scholar, 5Yao X. Wei D. Soden C., Jr. Summers M.F. Beckett D. Biochemistry. 1997; 36: 15089-15100Crossref PubMed Scopus (67) Google Scholar, 6Yao X. Soden C., Jr. Summers M.F. Beckett D. Protein Sci. 1999; 8: 307-317Crossref PubMed Scopus (26) Google had that deletion of residues at the amino or carboxyl of biotinoyl domain sequences in the of due to of domain J.E., Jr. J. Biol. Full Text PDF PubMed Google Scholar, J.E., Jr. 2000; PubMed Google Scholar). BCCP residues have these residues for deletion or the residues are from the of the domain protein and have been as of the linker region E.L. Shu N. Howard M.J. Broadhurst R.W. Chapman-Smith A. Wallace J.C. Morris T. Cronan J.E., Jr. Perham R.N. Biochemistry. 1999; 38: 5045-5053Crossref PubMed Scopus (69) Google Scholar). These residues are in the and have and for deletion or these residues are conserved in the BCCPs of J. 1993; PubMed Google Scholar) and D. Cronan J.E., Jr. D. J. 1995; PubMed Google both of which have been to the function of E. coli BCCP in vivo protein also a deletion within this of the BCCP residues upstream of residue with the biotinyl domain of the P. subunit J.E., Jr. J. Biol. Full Text Full Text PDF PubMed Scopus Google that these residues play a role in biotin domain structure. and in vivo function of a of and and BCCP function was to large whereas some small deletions gave or functional of the and deletions of and resulted in normal growth of at and 42 whereas a deletion of only failed to support The deletions and that the most defective in growth of lacked the sequence of residues located upstream of the biotinoyl region very to sequence deletions that the biotinoyl domain to an sequence an sequence or a all In contrast, deletions of upstream sequences as in the and BCCPs had or no The only to this was a deletion of in the sequences upstream of the biotinoyl domain of the mutant BCCP resulted in only growth of at of this is of the BCCP also growth at 42 that at failed to at the that an of activity in the biological due to of the proteins such as of the linker sequences. this of the was in strain at a nonpermissive the protein and with and the proteins by Expression of of the deleted BCCPs at in the of gave a of the some of these of the and data showed of biotinylated proteins in to the proteins with some the these proteins and the of protein due to was of no from of deleted and BCCP residues are by the and by The sequences the are the chimeric linker as and deletion The sequences the are the BCCP linker and deletion The first and BCCP residues are and is by at the J.E., Jr. J. Biol. Full Text Full Text PDF PubMed Scopus Google with to the only with a and no the grown at 30 in the of in a new residues are by the and by The sequences the are the chimeric linker as and deletion The sequences the are the BCCP linker and deletion The first and BCCP residues are and is by at the J.E., Jr. J. Biol. Full Text Full Text PDF PubMed Scopus Google with to the only with a and no the grown at 30 in the of and also the BCCP the with the chimeric protein, a deletion to the proline/alanine-rich sequence adjacent to the biotinoyl domain, was Expression of the protein gave only and of only of the 30 residues deleted in resulted in of most of the biological activity. these it the that the sequence play an role in this of biological activity. Therefore, this sequence was deleted from the linker to the protein with the that this protein biological activity. of the protein gave wild type growth the or of sequences adjacent to the biotinoyl domain to in BCCP in BCCP upstream proline/alanine-rich sequences for the deleted whereas in no sequences to act as of the deleted upstream residues for function of the BCCP, sequences adjacent to the biotinoyl domain of the protein to act as or the domain and the upstream proline/alanine-rich sequences. The sequences by from and these upstream of the biotinoyl domain of to the and of the protein growth at growth at 42 was of the protein, growth at was and growth also at 42 at a about of that upon of the wild type Therefore, the sequence of as an the biotin domain and the upstream sequences. that the proteins with these and in vivo of of the wild type BCCP sequence proteins or biotinylated. are of of the sequence with or gave proteins that and biotinylated. upon of the proteins growth of strain Therefore, residues of BCCP with very residues the of the biotinoyl domain as by the of and to in vivo A. Morris Wallace J.C. Cronan J.E., Jr. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google and the of biological activity to the of the a chimeric protein was in which the BCCP biotinoyl domain was with the only biotinoyl domain of that of P. D.V. Shenoy B.C. Carey P.R. Sonnichsen F.D. Biochemistry. 2000; 39: 2509-2516Crossref PubMed Scopus (48) Google Scholar). The fusion the proteins was a conserved residue such that the residue of the BCCP was with of the of this residue the residues of BCCP, and the residues of the subunit biotinoyl Expression of this chimeric protein failed to growth of mutant strain at nonpermissive although the chimeric protein was and efficiently biotinylated. Therefore, the conserved domain the biotinoyl domain of BCCP with a biotinoyl The proline/alanine-rich region of BCCP is essential for function of the protein as by the deletion The that a linker region from the subunit of the for the region that these protein segments play roles in the enzyme although the cognate have structures and the structures of the biotinoyl and lipoyl domains largely it that only the domain also the of the that these domains to their cognate proteins, have been the linker that of BCCP, only amino acid sequence is the BCCP and The of sequence is sequence is the that the E. coli lipoyl domains to the of the subunit is also only sequence the biotinoyl and lipoyl domains Perham R.N. Protein Sci. 1993; PubMed Scopus Google it is the structures the sequences of these protein domains that have been BCCP function is dependent the of the linker proteins having a deletion of residues an of residues a was deletions of some linker segments resulted in of function. Indeed, deletion within the BCCP linker had in vivo function in deletion of an subunit having a lipoyl domain Guest J.R. Perham R.N. PubMed Scopus Google Scholar, Guest J.R. Perham R.N. J. Mol. Biol. PubMed Scopus Google Scholar). In the of the linker residues had to deleted growth of an E. coli strain function of the subunit was strongly deletions had only The growth in of the enzyme activity, a of was Guest J.R. Perham R.N. J. Mol. Biol. PubMed Scopus Google Scholar). The most in these was upon of an linker for the growth of the E. coli strain and resulted in a of activity Guest J.R. Perham R.N. J. Mol. Biol. PubMed Scopus Google Scholar). such as of all residues or all proline residues resulted in that full although both of these flexible the Guest J.R. Protein 1993; PubMed Scopus Google Scholar). the of in the linker as as the weak the of a that the BCCP linker region or all of the linkers. this been In to for the when in the of BCCP, small deletions of the linker in of protein function in The deletion that the sequence of residues adjacent to the biotinoyl domain the most role of the linker in BCCP function. of the domain coli biotin protein ligase is by deletion of these residues and the proteins are in vivo, the in the function of BCCP in the having the sequence of the linker that was BCCP been by Laue E.D. Perham R.N. E. J. Biol. 264: Full Text PDF PubMed Google Scholar). the the and of a it as a of the sequences are in the whereas the for a is by the that the linker a of Laue E.D. Perham R.N. E. J. Biol. 264: Full Text PDF PubMed Google Scholar). the of proline and it is that the linker a flexible and that upon also to BCCP, the linker is a functional replacement for that of of the BCCP linker gave a in that deletion of the sequence adjacent to the domain had no protein function. of due to functional replacement of these residues with upstream sequences of sequences adjacent to the domain BCCP function. Therefore, the linker to sequences that the of upstream residues as of adjacent to the domain, whereas the BCCP linker lacks this linker and by the the structures of such protein segments is a of structural In of structural of the linker sequences in this was to the protein the at is to the of the for from sequence that of the BCCP biotinyl domain showed a with the domain in the of the linker residue in this the of in wild type The only in the of the linker as by the are no data which sequence or sequences for the of the and BCCP linkers. have been to the data in this with of activity. the of E. coli upon and the activity in P.R. The Scholar, P. E. J. J. Biol. Full Text PDF PubMed Google Scholar, J. Cronan Jr. J. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar).