Cloning, sequence analysis, and functional expression of the acetyl coenzyme A synthetase gene from Methanothrix soehngenii in Escherichia coli

Eggen, Rik I.L.; Geerling, A.C.M.; Boshoven, A.B.P.; Vos, Willem M. de

doi:10.1128/jb.173.20.6383-6389.1991

Cited by 68 publications

(31 citation statements)

References 37 publications

(28 reference statements)

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“…(29), the consensus sequence (SGTTGXPKG) in this region may represent a new class of phosphate-binding loops (60). The essential nature of this region has also been recognized by other investigators (16,29,46,61).…”

Section: Resultsmentioning

confidence: 94%

Biosynthesis of D-alanyl-lipoteichoic acid: cloning, nucleotide sequence, and expression of the Lactobacillus casei gene for the D-alanine-activating enzyme

Heaton

Neuhaus

1992

J Bacteriol

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The D-alanine-activating enzyme (Dae; EC 6.3.2.4) encoded by the dae gene from Lactobacius casei ATCC 7469 is a cytosolic protein essential for the formation of the D-alanyl esters of membrane-bound Upoteichoic acid. The gene has been cloned, sequenced, and expressed in Escherichia coli, an organism which does not possess Dae activity. The open reading frame is 1,518 nucleotides and codes for a protein of 55.867 kDa, a value in agreement with the 56 kDa obtained by electrophoresis. A putative promoter and ribosome-binding site immediately precede the dae gene. A second open reading frame contiguous with the doe gene has also been partially sequenced. The organization of these genetic elements suggests that more than one enzyme necessary for the biosynthesis of D-alanyl-lipoteichoic acid may be present in this operon. Analysis of the amino acid sequence deduced from the dae gene identified three regions with significant homology to proteins in the following groups of ATP-utilizing enzymes: (i) the acid-thiol ligases, (ii) the activating enzymes for the biosynthesis of enterobactin, and (iii) the synthetases for tyrocidine, gramicidin S, and penicillin. From these comparisons, a common motif (GXXGXPK) has been identified that is conserved in the 19 protein domains analyzed. This motif may represent the phosphate-binding loop of an ATP-binding site for this class of enzymes. A DNA fragment (1,568 nucleotides) containing the doe gene and its putative ribosome-binding site has been subcloned and expressed in E. coli. Approximately 0.5% ofthe total cell protein is active Dae, whereas 21% is in the form of inclusion bodies. The isolation of this minimal fragment without a native promoter sequence provides the basis for designing a genetic system for modulating the D-alanine ester content of lipoteichoic acid.The D-alanyl-ester content of lipoteichoic acid (LTA) appears to play a role in determining cell shape. Mutants that are deficient in these esters are characterized by aberrant morphology and defective cell separation (52). To establish further the role of these esters, genetic methods were sought for modulating their content in LTA.In Lactobacillus casei, the incorporation of D-alanine into ester-linked D-alanyl-LTA is accomplished in the following two-step reaction sequence (6,9,11,42,51,54): enzyme + D-alanine + ATP enzyme * AMP-D-alanine + PPi(1) enzyme * AMP-D-alanine + mLTA D-alanyl-mLTA + enzyme + AMP (2) In reaction 1, enzyme-bound aminoacyl adenylate is synthesized by the D-alanine-activating enzyme (Dae; EC 6.3.2.4) in the presence of ATP. In reaction 2, the activated D-alanine is covalently linked to membrane-associated LTA (mLTA) in the presence of D-alanine:membrane acceptor ligase (EC 6.3.2.16). The mechanism of LTA acylation catalyzed by the ligase has not been established.The D-alanyl esters of LTA have been implicated in at least three functions. These include the regulation of autolytic activity (22), the binding of Mg2+ for enzyme function (5,25,30,37), and the synthesis of wall teichoic acid (20...

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Section: Resultsmentioning

confidence: 94%

Biosynthesis of D-alanyl-lipoteichoic acid: cloning, nucleotide sequence, and expression of the Lactobacillus casei gene for the D-alanine-activating enzyme

Heaton

Neuhaus

1992

J Bacteriol

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“…Among the putative archaeal ACSs, the sequences of the Haloarcula marismortui ACS1 and P. aerophilum ACS (PA-ACS), for which enzymatic activities have been proven , reside in Groups VII and VIII, respectively (Figure 1). The Methanosaeta concilii ACS sequence in Group VII is identical to that of the Methanothrix soehngenii ACS, which has been purified and characterized (Jetten et al 1989, Eggen et al 1991.…”

Section: Phylogenetic Analysis Of Acsmentioning

confidence: 97%

“…Characterized enzymes from groups I, II, III, VI, and VII, which include the bacterial ACSs from S. enterica (Gulick et al 2003) and B. japonicum (Preston et al 1990, Lee et al 2001, the eukaryotic ACS1 and ACS2 enzymes from human 1.50 ± 0.02 19.0 ± 0.13 12.2 ± 0.09 (Luong et al 2000, Fujino et al 2001a) and S. cerevisiae (van den Berg et al 1996), and archaeal ACSs from M. concilii (M. soehngenii) (Jetten et al 1989, Eggen et al 1991, H. marismortui , and M. thermautotrophicum (MT-ACS1), all show a strong preference for acetate as the acyl substrate, with propionate being the only alternative acyl substrate. AF-ACS2 and the P. aerophilum PA-ACS , both from group VIII, have an expanded substrate range that includes butyrate and the branched chain isobutyrate as well as acetate and propionate.…”

Section: Discussionmentioning

confidence: 99%

AMP-forming acetyl-CoA synthetases in Archaea show unexpected diversity in substrate utilization

Ingram‐Smith

Smith

2006

Archaea

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Summary Adenosine monophosphate (AMP)-forming acetyl-CoA synthetase (ACS; acetate:CoA ligase (AMP-forming), EC 6.2.1.1) is a key enzyme for conversion of acetate to acetyl-CoA, an essential intermediate at the junction of anabolic and catabolic pathways. Phylogenetic analysis of putative short and medium chain acyl-CoA synthetase sequences indicates that the ACSs form a distinct clade from other acyl-CoA synthetases. Within this clade, the archaeal ACSs are not monophyletic and fall into three groups composed of both bacterial and archaeal sequences. Kinetic analysis of two archaeal enzymes, an ACS from Methanothermobacter thermautotrophicus (designated as MT-ACS1) and an ACS from Archaeoglobus fulgidus (designated as AF-ACS2), revealed that these enzymes have very different properties. MT-ACS1 has nearly 11-fold higher affinity and 14-fold higher catalytic efficiency with acetate than with propionate, a property shared by most ACSs. However, AF-ACS2 has only 2.3-fold higher affinity and catalytic efficiency with acetate than with propionate. This enzyme has an affinity for propionate that is almost identical to that of MT-ACS1 for acetate and nearly tenfold higher than the affinity of MT-ACS1 for propionate. Furthermore, MT-ACS1 is limited to acetate and propionate as acyl substrates, whereas AF-ACS2 can also utilize longer straight and branched chain acyl substrates. Phylogenetic analysis, sequence alignment and structural modeling suggest a molecular basis for the altered substrate preference and expanded substrate range of AF-ACS2 versus MT-ACS1.

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“…In the meantime, sequences of at least 25 peptide synthetases, comprising altogether 58 respective domains, and 38 other proteins have been obtained [1,3] and additional motifs described (A-I; Fig. 1) [1,[5][6][7][8]. This superfamily of enzymes and multienzymes has been subdivided according to structural similarities into four classes [2].…”

Section: Introductionmentioning

confidence: 99%

Expression of an active adenylate‐forming domain of peptide synthetases corresponding to acyl‐CoA‐synthetases

et al. 1995

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Peptide synthetases and acyl-CoA-synthetases form acyl adenylates which are transferred to CoA or enzyme-bound pantetheine. To verify the existence of an adenylate domain in peptide synthetases, a 60.8 kDa fragment of tyrocidine 1-synthetase was constructed by a 1629 bp deletion, expressed in Escherichia coli, and characterized. The truncated multienzyme activated phenylalanine and substrate analogues with comparable kinetics as the over-expressed synthetase, as judged by ATP-[32p]pp~ exchange reaction. Thus the N-terminal domain resembling an acyl-CoA-synthetase is an autonomous structural element. This N-terminal domain is followed by a cofactor binding domain, resembling acyl carrier proteins involved in polyketide formation.Key words." Tyrocidine synthetase; Multienzyme; Peptide synthetase; Acyl-CoA-synthetase; Pantetheine; CoA structed of modules, each contributing one amino-or hydroxy acid to the peptide structure. Each module contains an adenylate-forming domain, a cofactor binding site, and a condensation/modification domain. The adenylate forming domains display significant similarity to acyl-CoA-synthetases and related proteins. We have predicted, by alignment of sequences of these three classes and those of acyl carrier proteins also binding 4'-phosphopantetheine, the boundaries of adenylate and cofactor domains. The linker region is not rich in Ala, Pro and Glu residues, as in fatty acid synthases and 2-oxo-acid dehydrogenases containing similar cofactor domains. Still, amino acids frequently found in linker regions are dominating. Upon deletion of the cofactor and following functional domains of tyrocidine synthetase 1, a stable N-terminal fragment was obtained which catalysed adenylate formation with similar kinetic properties as the apo-form of the peptide synthetase [8].

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Cloning, sequence analysis, and functional expression of the acetyl coenzyme A synthetase gene from Methanothrix soehngenii in Escherichia coli

Cited by 68 publications

References 37 publications

Biosynthesis of D-alanyl-lipoteichoic acid: cloning, nucleotide sequence, and expression of the Lactobacillus casei gene for the D-alanine-activating enzyme

Biosynthesis of D-alanyl-lipoteichoic acid: cloning, nucleotide sequence, and expression of the Lactobacillus casei gene for the D-alanine-activating enzyme

AMP-forming acetyl-CoA synthetases in Archaea show unexpected diversity in substrate utilization

Expression of an active adenylate‐forming domain of peptide synthetases corresponding to acyl‐CoA‐synthetases

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