As of July 2009, there are 47 crystal structures deposited in the Protein Data Bank of members of the ANL adenylating enzyme family (). These structures represent 16 different proteins and have been crystallized in a variety of liganded states, providing a detailed view of the catalytic strategy used by this enzyme family.
Acyl-AMP forming adenylating enzymes that have been structurally characterized
The first crystal structure of a member of the adenylate-forming family of enzymes was of firefly luciferase from P. pyralis
). This structure identified an overall two-domain architecture with a larger N-terminal domain composed of the first 430 residues and a smaller C-terminal domain of the final 120 residues (). The larger domain contained an ababa domain structure with two large eight stranded β-sheets that surround two α-helices. The N-terminal domain ends with a distorted β-sheet. Following a short disordered loop in the luciferase structure, the C-terminal domain begins with an antiparallel β-sheet that contained two strands, followed by a central 3-stranded β-sheet that was surrounded by helices. The residues connecting the two domains form the A8 motif () and are collectively referred to as the A8 loop. Luciferase was crystallized in the absence of ligands. The conserved sequence motifs (40
) were used to propose a location of the enzyme active site. In particular, the Gly- and Ser-rich motif I was located at the interface between the N- and C-terminal domains. Conti et al. (39
) note that the cleft is likely “too big to accommodate the substrates” and predict closure of the interface upon substrate binding.
Crystal Structures of A. Firefly Luciferase and B. PheA adenylation domain
As noted above, early experiments predicted a large conformational change for acyl-CoA synthetases and luciferase (1
) on the basis of tritium exchange and thermal inactivation in the presence and absence of ligands. A conformational change was also invoked to explain the stabilization of phenylacetyl-CoA ligase (41
). The crystal structure of firefly luciferase thus provided a structural framework to envision this large conformational change. The single structure, however, provided only an initial look at the protein and many additional structural studies were necessary to understand the full conformational mechanism.
The structure of an initiating NRPS adenylation domain, the phenylalanine activating domain of gramicidin synthetase S (GrsA), was determined the following year (42
). Importantly, this structure was determined in the presence of the amino acyl substrate phenylalanine and a molecule of AMP () confirming the predicted location of the active site. The C-terminal domain was rotated by ~90° compared to the orientation seen in the luciferase structure. A universally conserved lysine from the A10 region formed hydrogen bonds to the ribose ring oxygen, the 5′-bridging oxygen, and a carboxylate oxygen of phenylalanine. This suggested a possible catalytic role for this lysine, which was indeed supported by prior biochemical studies of tyrocidin synthetase (40
These initial structures provided the foundation for a number of studies that investigated the roles of catalytic or substrate specificity residues. In particular, the interaction of the C-terminal lysine from the A10 region () was studied by mutagenesis in both luciferase (43
) and PrpE, a propionyl-CoA synthetase (44
). No activity could be detected for the complete reaction of the K592E mutant of PrpE for example and activity was reduced by over four orders of magnitude for the reverse of the adenylation reaction; the rate of production of propionyl-CoA from the propionyl-AMP intermediate, however, was reduced by only a factor of 2. These studies demonstrated a role of this residue specifically in the adenylation partial reaction.
Subsequent to the structural characterization of PheA, a number of additional structures of enzymes in this family were determined that demonstrated a similar tertiary structure and a similar conformational orientation between the N- and C-terminal domains. These structures included DhbE, the self standing adenylation domain from the bacillibactin NRPS cluster (45
), yeast acetyl-CoA synthetase (46
), two enzymes that catalyze aryl-CoA synthesis that are involved in the metabolic breakdown of 4-chlorobenzoate (47
) and benzoic acid (48
), luciferase from L. cruciola
), and the enzyme DltA from B. cereus
, which is involved in the activation of alanine for subsequent alanylation of teichoic acid in cell wall biosynthesis of gram positive bacteria (50
). These structures were all determined in the absence of ligands, or in the presence of the acyl substrate, the adenylate, or AMP. Notably, none of these structures contained a bound CoA or thiol acceptor for the second partial reaction ().
Insights into CoA binding were derived from the crystallization of a bacterial acetyl-CoA synthetase (Acs) bound to adenosine-5′-propylphosphate, a non-hydrolyzable mimic of the adenylate intermediate, and CoA (52
). This structure, which appeared to show the enzyme poised to catalyze the thioesterification reaction, located the nucleotide of CoA at the surface of the protein with the pantetheine portion of CoA passing through a pantetheine tunnel
that runs between the N- and C-terminal domains to enter the mostly buried adenylate binding site.
The most intriguing feature of this structure was that the C-terminal domain of Acs adopted a dramatically different conformation compared to that seen in the prior structures of PheA and DhbE (). The C-terminal domain of Acs packed against the N-terminal domain forming a more lobular enzyme (). Multiple interactions were observed between the two domains, and between the C-terminal domain and the reaction intermediates. In this conformation, the loop containing the A10 lysine residue is 25Å from the active site. In contrast, the A8 β-sheet that initiates the C-terminal domain was rotated into the active site. The luciferase C-terminal domain, which was also observed in a conformation than observed in PheA, did not make any interactions with the N-terminal domain and did not seem to be a functionally relevant conformation.
Crystallographic Structure of Acetyl-CoA synthetase
The biochemical data implicating the A10 lysine from the C-terminal domain in catalyzing the adenylate-forming reaction specifically (43
) and the structural observation of this new conformational state of Acs bound to CoA provided preliminary support for a novel catalytic strategy (52
). In this proposed catalytic strategy, the members of this adenylate-forming family would adopt the PheA-like structure to catalyze the adenylation partial reaction. Upon formation of the adenylate and the release of pyrophosphate, the C-terminal domain would rotate to the orientation observed in the bacterial Acs to form a second conformation that would be used to catalyze the thioester-forming reaction. We adopted the term Domain Alternation
, which had been used to describe a large-scale domain rearrangement in methionine synthase (53
), to describe this mechanism.
The crystal structures of several other members of this enzyme family have since been determined in this conformation. These structures include the long chain fatty acyl-CoA synthetase from T. thermophilus
), DltA from B. subtilis
), and an acyl-adenylating enzyme from the methanogenic bacteria Methanosarcina acetivorans
). Interestingly, while the Acs structure contained CoA, these latter structures did not contain bound coenzyme A, although in the case of the fatty acyl-CoA synthetase, CoA was included in the crystallization conditions yet was not bound in the crystal structure.
In the last year, two examples of a single enzyme being crystallized in both conformations have been reported. The 4-chlorobenzoyl-CoA ligase (4CBL) from Alcaligenes
sp. AL3007 was the subject of extensive structural and kinetic evaluation, as will be discussed below. As part of this study, the enzyme was trapped in both the adenylate-forming conformation bound to the adenylate intermediate as well as to a complex of AMP and the product analog 4-chlorophenacyl-CoA (57
). More recently, the structure of the human medium chain Acyl-CoA synthetase has also been determined in both conformational states (58
). These alternate structures demonstrate that the domain rotation is a dynamic feature of the ANL enzymes and does not simply reflect differences in tertiary organization between different superfamily members.
Not surprisingly, crystallization of these conformationally flexible enzymes has been challenging and several of the deposited structures exhibit significant disorder in the C-terminal domain. Indeed, a recent structure of an FAAL (34
) required the removal of the C-terminal domain altogether to achieve crystallization. As with all structural studies, the careful selection of appropriate inhibitors can support the crystallization of enzymes trapped in relevant conformations. The use of alkyl phosphate esters (52
) or adenosyl sulfamate analogs (49
) as mimics of the adenylate intermediate, or a substituted phenacyl-CoA thioether (57
) as a mimic of the CoA thioester product has enabled the determination of some of the highest resolution structures of members of this conformationally dynamic enzyme family.