The reductase domain in a Type I fatty acid synthase from the apicomplexan Cryptosporidium parvum: Restricted substrate preference towards very long chain fatty acyl thioesters
© Zhu et al; licensee BioMed Central Ltd. 2010
Received: 22 August 2010
Accepted: 22 November 2010
Published: 22 November 2010
The apicomplexan Cryptosporidium parvum genome possesses a 25-kb intronless open reading frame (ORF) that predicts a multifunctional Type I fatty acid synthase (CpFAS1) with at least 21 enzymatic domains. Although the architecture of CpFAS1 resembles those of bacterial polyketide synthases (PKSs), this megasynthase is predicted to function as a fatty acyl elongase as our earlier studies have indicated that the N-terminal loading unit (acyl-[ACP] ligase) prefers using intermediate to long chain fatty acids as substrates, and each of the three internal elongation modules contains a complete set of enzymes to produce a saturated fatty acyl chain. Although the activities of almost all domains were confirmed using recombinant proteins, that of the C-terminal reductase domain (CpFAS1-R) was yet undetermined. In fact, there were no published studies to report the kinetic features of any reductase domains in bacterial PKSs using purified recombinant or native proteins.
In the present study, the identity of CpFAS1-R as a reductase is confirmed by in silico analysis on sequence similarity and characteristic motifs. Phylogenetic analysis based on the R-domains supports a previous notion on the bacterial origin of apicomplexan Type I FAS/PKS genes. We also developed a novel assay using fatty acyl-CoAs as substrates, and determined that CpFAS1-R could only utilize very long chain fatty acyl-CoAs as substrates (i.e., with activity on C26 > C24 > C22 > C20, but no activity on C18 and C16). It was capable of using both NADPH and NADH as electron donors, but prefers NADPH to NADH. The activity of CpFAS1-R displayed allosteric kinetics towards C26 hexacosanoyl CoA as a substrate (h= 2.0; Vmax = 32.8 nmol min-1 mg-1 protein; and K50 = 0.91 mM).
We have confirmed the activity of CpFAS1-R by directly assaying its substrate preference and kinetic parameters, which is for the first time for a Type I FAS, PKS or non-ribosomal peptide synthase (NRPS) reductase domain. The restricted substrate preference towards very long chain fatty acyl thioesters may be an important feature for this megasynthase to avoid the release of product(s) with undesired lengths.
Cryptosporidium is a group of important parasites that infect a wide range of hosts from reptiles and birds to humans and other mammals [1–3]. Among them, C. parvum is zoonotic and infects both humans and animals. Cryptosporidium infection in immunocompetent individuals may cause self-limiting diarrhea, but its infection in immunocompromized patients can be chronic and life-threatening [4, 5]. Therefore, it is an important opportunistic pathogens in AIDS patients. Additionally, there are no effective treatments against cryptosporidial infection in AIDS patients.
Due to the extreme large size of CpFAS1, it is impractical to express the entire megasynthase for biochemical analysis. Therefore, we have adapted a "divide and conquer" strategy and successfully expressed individual units/modules to study their biochemical features . Our previous experiments using recombinant proteins have shown that the CpFAS1 loading unit has a substrate preference to medium and long chain fatty acids (MCFAs and LCFAs), and all domains within the three internal modules are enzymatically functional, suggesting that CpFAS1 is likely involved in the elongation of saturated fatty acid(s), rather than the de novo fatty acid synthesis [12, 13]. However, although the C-terminal R domain was expressed, its biochemical features were yet to be determined then due to the lack of an appropriate assay.
Here we report our recently developed assay and experiments that reveal some unique biochemical features for the CpFAS1 acyl reductase domain (CpFAS1-R) including the substrate preference and kinetics for the first time for this family of acyl reductases. We have not only confirmed the reductive activity of CpFAS1-R, but also determined that it could only utilize very long chain fatty acyl thioesters as its substrates. Additionally, we have also performed phylogenetic analysis and observed bacterial-affinity of reductase domains from various apicomplexan FASs and PKSs.
Sequence features and evolutionary affiliation of the CpFAS1-R domain
Among apicomplexans, Type I FAS/PKS genes are only discovered from Cryptosporidium and cyst-forming and intestinal coccidia (eg. Toxoplasma and Eimeria), but absent in the hematozoa that contain only Type II FAS (eg. Plasmodium and Babesia) or no FAS at all (eg. Theileria) [7, 12, 14–16]. Both Cryptosporidium and Toxoplasma genomes contain reductase domains at the C-terminal ends of the Type I FAS and PKS, respectively. The E. tenella genome is only partially sequenced (see http://www.sanger.ac.uk/Projects/E_tenella/), from which only one reductase domain could be recovered. The architectures of CpFAS1 and CpPKS1 resemble bacterial and fungal PKS rather than the Type I FAS in humans and animals . Additionally, these parasite megasynthases appear to use acyl reductase domains to release the fatty acyl or polyketide chains as "long chain" alcohols, rather than using thioesterase (TE) to produce acids (Figure 1B). However, none of the apicomplexan genomes encodes any non-ribosomal peptide synthase (NRPS) that may act alone or together with PKSs to produce complex products.
Like most acyl reductase domains of the type I PKS and NRPS proteins, CpFAS1-R is located at the C-terminus and is defined by approximately 780 amino acids (aa) (Figure 1A). The first half of the domain contains conserved sequences and motifs characteristic to acyl reductase domains of PKSs and NRPSs, and to the fungal L-aminoadipate-semialdehyde dehydrogenase (AASDH), which include the Rossmann-fold NAD(P)H binding site "Gx1-2GxxG" and a reduction motif "GYxxSKWxxE" (Figure 1) [17–20]. However, the C-terminal half of the domain appears to be unique to the apicomplexans, as it is only homologous to the other apicomplexan FAS1 and PKS1 R domains, but not to any other species in the databases, nor does it contain any putative motifs.
Sequence comparisons indicated that the CpFAS1-R domain was more closely related to those of bacterial or fungal PKS/NRPS proteins. When CpFAS1-R domain was used as a query to search animal protein databases, there were no hits from vertebrate sequences. The only hits with significantly high identities are three invertebrate proteins annotated as oxidoreductase family or hypothetical proteins, i.e., Ciona intestinalis, (XP_002121624, E-value = 2.E-25), Brugia malayi (XP_001899674, 9.E-25), and Trichoplax adhaerens (XP_002112422, 8.E-15). Other invertebrate hits with much lower identities were a number of acyl-CoA reductases, mainly from insects such as Drosophila mojavensis (XP_002000602, 4.E-10) and Culex quinquefasciatus (XP_001847721, 1.E-9). This is in contrast to the hits from bacterial and fungal proteins, in which >420 and >170 hits displayed E-values at or smaller than 9.E-10 and 9.E-20, respectively.
With the exception for CpPKS1-R, all apicomplexan FAS/PKS reductase domains (including CpFAS1-R) formed a single cluster that is fully supported by PP value (Figure 2, red branches). This group is then clustered together with several bacterial PKS/NRPS reductases. However, the CpPKS1-R domain alone was unexpectedly placed within another bacterial PKS/NRPS cluster. Although this observation is of some implication that CpFAS1 and CpPKS1 (or their R domains) might have different evolutionary origins, more rigorous analyses using additional domains are needed to truly make firm conclusions. Nonetheless, these observations support a bacterial affiliation of apicomplexan FAS/PKS R domains, as previously observed for the acyl transferase (AT) domains from CpFAS1 and CpPKS1 .
CpFAS1-R domain's substrate preference
The R domain-catalyzed release of final products by bacterial and fungal PKS/NRPS polypeptides were mainly determined by analyzing the products in native or heterogeneous hosts expressing engineered PKS/NRPS genes . There were several studies that directly observed the release/formation of products using recombinant PKS/NRPS modules [21–23]. However, there were no reported studies to directly assay the R domain activities and measure the kinetic parameters in vitro. On the other hand, it is impractical (if not impossible) to obtain sufficient pure Cryptosporidium materials for extensive biochemical analyses, and molecular tools are also unavailable to genetically manipulate this parasite. Therefore, heterogeneous expression systems and the use of recombinant proteins may be the only approaches currently available to study the biochemical feature of the CpFAS1 or CpPKS1 proteins and domains.
Suitable substrates are required to directly assay the R domain activity. However, there are some technical difficulties in preparing the native substrates for CpFAS1-R (i.e., very long chain fatty acyl-ACP). On the other hand, we have previously observed that the N-terminal AL domains from CpFAS1 and CpPKS1 could utilize LCFAs and CoA as substrates to form fatty acyl-CoA [13, 24], and additionally, the CpFAS1-R domain is related to the acyl-CoA reductases (Figure 2). This prompted us to test whether fatty acyl-CoAs could also be used by CpFAS1-R as substrates to assay its activity.
As mentioned above, we have previously determined that the CpFAS1-AL domain prefers LCFAs as its substrates . This may act as the first checkpoint to ensure correct fatty acids are loaded into this megasynthase for elongation. In this study, we have observed that CpFAS1-R domain could only utilize VLC fatty acyl-CoA, indicating that this R domain may act as a second checkpoint to ensure that correct lengths of products can be released.
The PKS/NRPS R domains may catalyze a single-step reduction (two-electron transfer) to produce fatty aldehyde, or a two-step reduction (four-electron transfer) to produce fatty alcohol . Therefore, it is yet unclear whether CpFAS1-R-released products are VLC fatty aldehydes or fatty alcohols. We have repeatedly attempted, but failed to detect the CpFAS1-R-released acyl chains using thin layer chromatography (TLC) and mass spectrometry. On the other hand, TLC was able to distinguish C16 fatty alcohol and aldehyde using standards or preparations in controlled experiments (data not shown), or by other investigators . It is possible that the limited amounts of VLC fatty acyl products were likely precipitated or aggregated after being separated from CoA to become insoluble for TLC or MS detection. However, since aldehydes are toxic to cells, and no additional VLC fatty acyl reductases are present in the Cryptosporidium genomes, we speculate that the final products produced by CpFAS1 are likely VLC fatty alcohols. Nonetheless, based on previously reported data and the present study, we are able to propose a general scheme of reactions catalyzed by the various domains of CpFAS1 as shown in Figure 1B.
Since the giant CpFAS1 and CpPKS1 are structurally and functionally different from humans Type I FAS, these parasite megasynthases may serve as novel drug targets. CpFAS1 and CpPKS1 also utilize R domains to release final products, which differs from human FAS that uses a thioesterase (TE) to product fatty acids. This unique feature suggests that like several other enzymatic domains, the reductase domains in CpFAS1 and CpPKS1 may also be explored as drug targets. The production of recombinant CpFAS1-R protein together with the novel assay developed in this study make it possible for the screening of inhibitors for potential drug development.
The CpFAS1-R domain protein sequence was used as a query to repeatedly search the NCBI genome and protein databases with various BLAST algorithms. More than 150 hits with e-values < 10-20 were retrieved, which included discrete enzymes or reductase domains of multi-functional proteins. Multiple alignments were performed using a unix-based MUSCLE program (version 3.6), and short and nearly identical sequences were removed from the dataset. The dataset was further refined by additional multiple alignments with MUSCLE [26, 27], and sampling of representative taxa to produce a final dataset containing 91 taxa and 184 aa positions. Conserved domains and motifs were identified and visualized as sequence logos by bits with the height of symbols within the stack indicates the relative frequency of each amino of the positions using WebLogo 3 (http://weblogo.threeplusone.com/) .
Bayesian inference (BI)-based phylogenetic reconstructions were performed with a parallel version of MrBayes program (version 3.1.2; http://mrbayes.csit.fsu.edu/) ). A WAG amino acid substitution model was used in the BI analysis. Among-site rate heterogeneity considered the fraction of invariance (Finv) and a discrete 12-rate gamma distribution (i.e., WAG + Finv + Γ(12)). At least 106 generation of searches were performed with two independent runs, each containing four chains running simultaneously. The current trees were saved every 100 generations, and the posterior probability (PP) values were calculated after the first 25% trees were discarded. The final consensus tree was visualized using a FigTree program (version 1.3.1; http://tree.bio.ed.ac.uk/software/figtree/).
where Y is the enzyme activity, [S] is the substrate concentration, K50 is the concentration of S to achieve 50% Vmax value (similar to Km), and h is the Hill slope.
fatty acid synthase
long chain fatty acid
medium chain fatty acid
non-ribosomal peptide synthase
very long chain fatty acid
open reading frame
This research was supported by a Grant (R01 AI44594) from the National Institutes of Health (NIH) under the United States Department of Health and Human Services (DHHS).
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