The acyl-adenylate-forming enzyme superfamily, consisting of acyl- and aryl-CoA synthetases, the adenylation domain of the nonribosomal peptide synthetases, and luciferase, has three signature motifs (I–III) and ten conserved core motifs (A1–A10), some of which overlap the signature motifs. The consensus sequence for signature motif III (core motif A7) in acetyl-CoA synthetase is Y-X-S/T/A-G-D, with an invariant fifth position, highly conserved first and fourth positions, and variable second and third positions. Kinetic studies of enzyme variants revealed that an alteration at any position resulted in a strong decrease in the catalytic rate, although the most deleterious effects were observed when the first or fifth positions were changed. Structural modeling suggests that the highly conserved Tyr in the first position plays a key role in active site architecture through interaction with a highly conserved active-site Gln, and the invariant Asp in the fifth position plays a critical role in ATP binding and catalysis through interaction with the 2′- and 3′-OH groups of the ribose moiety. Interactions between these Asp and ATP are observed in all structures available for members of the superfamily, consistent with a critical role in substrate binding and catalysis for this invariant residue.
AMP-forming acetyl-CoA synthetase (Acs, EC 6.2.1.1), which catalyzes the formation of acetyl-CoA from acetate, ATP, and CoASH (acetate + ATP + CoASH
Sequence alignment of members of this superfamily has revealed the presence of three signature motifs as defined by Chang et al. [ motif I: motif II: motif III:
(boldfaced residues are the predominant residue at each position, and alternative residues are indicated in bracket.)
Marahiel et al. [
Here, we have investigated the role of motif III in acetyl-CoA synthetase (Acs) in the
Chemicals were purchased from VWR Scientific Products, Fisher Scientific, or Sigma Chemicals. Oligonucleotides for site-directed mutagenesis were purchased from Integrated DNA Technologies. IRD-700- and IRD-800-labeled oligonucleotides for DNA sequencing were purchased from Li-Cor Biosciences or MWG Biotech.
Sequence alignments were performed using Clustal X [
Site-directed mutagenesis of the gene-encoding
Unaltered Acs1Mt and its variants were heterologously produced in
The variants were subjected to gel filtration chromatography to determine subunit composition. A Superose 12 gel filtration column (GE Healthcare), preequilibrated with 50 mM Tris [pH 7.5] containing 150 mM KCl, was calibrated with chymotrypsinogen (25 kDa), ovalbumin (43 kDa), albumin (67 kDa), aldolase (158 kDa), catalase (232 kDa), ferritin (440 kDa), and blue dextran (2000 kDa).
Enzymatic activity was determined by the hydroxamate assay, which monitors formation of activated acyl groups such as the acetyl-CoA product of the ACS reaction [
For determination of apparent kinetic parameters, one substrate was varied, and the other two substrates were held at a saturating concentration, generally ten times the
Inhibition of wild-type Acs1Mt by adenosine and its derivatives and ribose was determined using the hydroxamate assay. In these assays, all three substrates were held at saturating levels, and ribose or adenosine was added to the reaction mix to a final concentration ranging from 0 to 1000 mM or 0 to 100 mM, respectively. The
The structures of Acs1Mt and the variants were modeled on the
Sequence alignment indicates considerable conservation of motif III among the Acs sequences, with the first, fourth, and fifth positions highly or completely conserved, but the second and third positions showing a higher level of variability and an overall consensus of Y-X-S/T/A-G-D (Table
Alignment of motif III residues in Acs sequences.
Consensus |
|
||
|
498 |
|
503 |
|
496 |
|
501 |
|
555 |
|
560 |
|
525 |
|
530 |
|
545 |
|
550 |
|
515 |
|
520 |
|
509 |
|
514 |
|
514 |
|
519 |
|
442 | WLL |
446 |
|
442 | WLL |
446 |
|
379 |
|
383 |
* * |
Positions within each sequence are shown, and conserved residues are indicated with an asterisk.
Inspection of the AcsSe and AcsSc structures places motif III in the active site, regardless of which conformation of the enzyme. The positioning of motif III residues near the adenylate moiety of the bound AMP ligand suggests that residues in this motif may play a role in ATP binding and/or catalysis. Modeling of Acs1Mt on the
Position of motif III residues in the active sites of (a) AcsSc, (b) AcsSe, and (c) the Acs1Mt structural model. Acs1Mt modeled on the AcsSe structure (PDB:2P2F) using Accelrys DS Modeler and the stereo images were created using Accelrys DS ViewerPro 5.0. For clarity in viewing, only some residues within the 10Å sphere of AMP are shown. AMP is shown in blue, and the motif III residues are shown in green. Residues discussed in the text are labeled.
In Acs1Mt, motif III has the sequence 498YTAGD502. We individually altered each position of motif III in Acs1Mt and determined the kinetic parameters of the purified enzyme variants. The residues in the highly conserved first, fourth, and fifth positions were changed to either Ala or a conservative amino acid replacement. The Thr residue at the more variable second position was changed to Ala, and the Ala residue in the third position was changed to Thr, as this is the residue found in many Acs sequences. The kinetic parameters for the purified enzyme variants were determined using the hydroxamate assay and are shown in Table
Kinetic parameters for ACS1Mt wild-type and variant enzymes.
Enzyme |
|
|
|
|
---|---|---|---|---|
(sec−1) | (mM) | (mM) | (mM) | |
Acs1Mt | 66.6 ± 0.9 | 3.5 ± 0.1 | 3.3 ± 0.2 | 0.19 ± 0.003 |
Tyr498Ala | 1.6 ± 0.04 | 7.5 ± 0.6 | 1.7 ± 0.3 | 0.10 ± 0.004 |
Tyr498Phe | Inactivea | |||
Thr499Ala | 0.8 ± 0.01 | 3.0 ± 0.01 | 1.7 ± 0.1 | 0.24 ± 0.01 |
Ala500Thr | 1.5 ± 0.04 | 2.1 ± 0.2 | 3.6 ± 0.1 | 0.51 ± 0.04 |
Gly501Ala | 0.3 ± 0.01 | 1.4 ± 0.1 | 1.7 ± 0.07 | 0.08 ± 0.002 |
Asp502Ala | Inactivea | |||
Asp502Glu | Inactivea | |||
Asp502Asn | Inactivea |
aActivity was tested over a wide range of concentrations for each substrate and at several enzyme concentrations, but no activity was observed.
The hydroxamate assay measures activated acyl groups including both acetyl-AMP and acetyl-CoA by their conversion to the acyl hydroxamate and subsequently to a ferric hydroxamate complex. Wilson and Aldrich [
No activity was detected with Acs1Mt with acetate in the absence of HSCoA, indicating that the acetyl-AMP intermediate remains enzyme bound and that the bound intermediate is not reactive with hydroxylamine. Thus, the kinetic parameters shown in Table
Several of the variants were found to be inactive over a wide range of concentrations for each substrate and a range of enzyme concentrations. Enzymes that were inactive displayed similar behavior in both the ion exchange and hydrophobic interaction chromatography steps during purification, and gel filtration chromatography indicated the variants are dimeric as for the wild type enzyme, suggesting there are no gross structural alterations. Overall, alteration of any of the residues in motif III appeared to have a strong deleterious effect on catalysis, although substrate affinity was generally not impaired.
Based on the two Acs structures, the highly conserved Tyr498 in the first position of motif III is part of a hydrogen bond network with Gln417 and through this hydrogen bond network may contribute to maintenance of the active-site architecture near the ATP binding site (Figures
The second and third positions of motif III, represented by Thr499 and Ala500 in Acs1Mt, are less well conserved than the other positions (Table
These positions were individually altered to Ala and Thr, respectively, in Acs1Mt, and the purified variants were analyzed. The
Gly501 in the fourth position of motif III is almost completely conserved within the acyl-adenylate-forming enzyme superfamily except for a few members most distantly related to Acs. Replacement of this residue by Ala resulted in two- to threefold reduced
To investigate the role of the invariant Asp residue of motif III, Asp502 of Acs1Mt was altered to Ala and the more conservative residues Glu and Asn. Although the enzyme variants were soluble, each of these alterations eliminated all detectable enzymatic activity, regardless of substrate concentrations or concentration of enzyme used. The fact that even the most conservative changes inactivated the enzyme indicates that this Asp is absolutely critical for activity, as might be expected since Asp502 is completely conserved among all ACSs and throughout the superfamily.
Inhibition assays were performed as an indirect approach to delineate the interaction between Asp502 and ATP. Ribose completely inhibited enzyme activity at concentrations above 600 mM, and the
Inhibition of Acs1Mt. (a) Ribose, (b) adenosine, (c) 2′-deoxyadenosine, and (d) 3′-deoxyadenosine. Assays were performed with the indicated concentrations of inhibitor in the reaction, and results are plotted as a percentage of the activity observed in the absence of inhibitor, with error bars as shown. The
To determine more precisely the interaction between Asp502 and the 2′- and 3′-OH groups of the ribose sugar of adenosine, inhibition by 2′- and 3′-deoxyadenosine was examined. Although only partial inhibition was observed with either compound, extrapolation of the results gave apparent
The Acs1Mt model (Figure
The active-site architecture in AcsSc and AcsSe is similar in the vicinity of the AMP ligand (Figures
Structures have been determined for a number of enzymes spanning the adenylate-forming enzyme superfamily, including short, medium, and long-chain acyl-CoA synthetases, the aryl-CoA synthetases CBL and benzoyl-CoA ligase, several NRPS adenylation domains, and luciferase. These structures have revealed that domain alternation between the first and second steps of the reaction is universal among the superfamily [
Three other members of the superfamily have structures in both the adenylate-forming and thioester-forming conformations. In 4-chlorobenzoate:CoA ligase (CBL), Asp385 hydrogen bonds with just the 2′-OH, whereas the 3′-OH interacts with Arg400 in the adenylate-forming conformation, whereas in the thioester-forming conformation, Asp385 maintains its hydrogen bond with the 2′-OH and now also interacts with 3′-OH group along with Arg400 [
The role of this invariant Asp has been studied biochemically in only a few members of the superfamily. In 3-chlorobenzoate-CoA ligase, alteration of this Asp to Val essentially eliminated all catalytic activity [
In CBL, the invariant Asp385 of motif III hydrogen bonds with the 2′-OH group. Alteration of this residue to Ala greatly reduced the overall rate of catalysis, primarily due to a reduced rate for the first step of the reaction, and resulted in increased
The results from this investigation indicate that the motif III/A7 signature motif in Acs plays an important role in both active-site architecture and ATP binding and catalysis.
Although all positions of this motif appear to play an important role in catalysis, the Tyr at the first position that is highly conserved among Acs sequences helps maintain active site architecture through a hydrogen bond network with other active-site residues, particularly the well-conserved Gln in motif II/A5 (Gln417 of Acs1Mt). Asp at the last position plays a critical role in active-site architecture through a hydrogen bond network and in ATP binding and catalysis through key interactions with the hydroxyl groups of the ribose moiety. This Asp is invariant across the entire superfamily, consistent with a critical role in ATP binding and catalysis of the adenylate-forming first step of the reaction in all members.
C. I.-Smith and J. L. Thurman Jr. contributed equally to this work.
Financial support for this project was provided by NIH (Award GM69374-01A1), the South Carolina Experiment Station (Project SC-1700198), and Clemson University. This paper is Technical Contribution no. 6040 of the Clemson University Experiment Station.