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

Guan Zhu,Xiangyu Shi,Xiaomin Cai

doi:10.1186/1471-2091-11-46

Abstract

BackgroundThe 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.ResultsIn 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).ConclusionsWe 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.

Highlights

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
Sequence features and evolutionary affiliation of the CpFAS1-R domain Among apicomplexans, Type I FAS/polyketide synthase (PKS) genes are only discovered from Cryptosporidium and cyst-forming and intestinal coccidia
Considering that the final CpFAS1 product(s) could be at least 6-carbons longer than the medium chain fatty acid (MCFA) or long chain fatty acid (LCFA) loaded by the AL domain [12,13], we extended our analysis to include a wide range of all commercially available long chain to very long chain fatty acyl-CoAs, and eventually observed activities

Summary

Introduction

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. As a group of early branch apicomplexans, Cryptosporidium lacks an apicoplast and its associated metabolic pathways (e.g., isoprenoid and Type II fatty acid synthetic pathways). It has virtually lost the capacity to synthesize any nutrients de novo [6,7,8,9]. The AL domain loads a fatty acid to the ACP to form an acyl-ACP, in which the acyl chain may be elongated by the internal modules and released by the R domain (Figure 1B)

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