Expression and functional characterization of a giant Type I fatty acid synthase (CpFAS1) gene from Cryptosporidium parvum

Zhu, Guangxi; Li, Yanan; Cai, Xiwen; Millership, Jason J.; Marchewka, Mary J.; Keithly, Janet S.

doi:10.1016/j.molbiopara.2003.11.011

Cited by 49 publications

(60 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Among these two C. parvum megasynthases, preliminary biochemical analysis using recombinant proteins has indicated that CpFAS1 may be involved in the elongation, rather than the de novo synthesis, of fatty acids (Zhu, 2004;Zhu et al, 2004). However, nothing was previously known about the biochemical features of CpPKS1.…”

Section: Discussionmentioning

confidence: 99%

Functional characterization of the acyl-[acyl carrier protein] ligase in the Cryptosporidium parvum giant polyketide synthase

Fritzler

Zhu

2007

International Journal for Parasitology

Self Cite

View full text Add to dashboard Cite

The apicomplexan Cryptosporidium parvum possesses a unique 1,500-kDa polyketide synthase (CpPKS1) comprised of 29 enzymes for synthesizing a yet undetermined polyketide. This study focuses on the biochemical characterization of the 845-amino acid loading unit containing acyl-[ACP] ligase (AL) and acyl carrier protein (ACP). The CpPKS1-AL domain has a substrate preference for long chain fatty acids, particularly for the C20:0 arachidic acid. When using [ 3 H] palmitic acid and CoA as co-substrates, the AL domain displayed allosteric kinetics towards palmitic acid (Hill coefficient, h = 1.46, K 50 = 0.751 μM, V max = 2.236 μmol mg −1 min −1 ) and CoA (h = 0.704, K 50 = 5.627 μM, V max = 0.557 μmol mg −1 min −1 ), and biphasic kinetics towards adenosine 5′-triphosphate (K m1 = 3.149 μM, V max1 = 373.3 nmol mg −1 min −1 , K m2 = 121.0 μM, and V max2 = 563.7 nmol mg −1 min −1 ). The AL domain is Mg 2+ -dependent and its activity could be inhibited by triacsin C (IC 50 = 6.64 μM). Furthermore, the ACP domain within the loading unit could be activated by the C. parvum surfactin production element-type phosphopantetheinyl transferase. After attachment of the fatty acid substrate to the AL domain for conversion into the fatty-acyl intermediate, the AL domain is able to transfer palmitic acid to the activated holo-ACP in vitro. These observations ultimately validate the function of the CpPKS1-AL-ACP unit, and make it possible to further dissect the function of this megasynthase using recombinant proteins in a stepwise procedure.

show abstract

Section: Discussionmentioning

confidence: 99%

Functional characterization of the acyl-[acyl carrier protein] ligase in the Cryptosporidium parvum giant polyketide synthase

Fritzler

Zhu

2007

International Journal for Parasitology

Self Cite

View full text Add to dashboard Cite

show abstract

“…The ACP domains within the loading unit (CpACP1) and elongation module 1 (CpACP2) of CpFAS1 were engineered into the pET24a (Novagen) and pMAL-c2x (New England Biolabs) vectors, respectively, as previously reported (44). The expression of T7-tag-fused CpACP1 (T7-CpACP1) or maltose-binding protein (MBP)-fused CpACP2 (MBP-CpACP2) was carried out in E. coli Rosetta (DE3) or Rosetta strains.…”

Section: Methodsmentioning

confidence: 99%

“…Fatty acid synthesis has been recently discovered for the phylum Apicomplexa, which contains many important protozoon parasites (e.g., Plasmodium, Babesia, Toxoplasma, Sarcosystis, Cyclospora, Eimeria, and Cryptosporidium) (32,40,42,44,45). As a reflection of their divergent parasitic life styles, the fatty acid metabolism is also diverse among apicomplexans.…”

mentioning

confidence: 99%

“…However, Cryptosporidium parvum employs only a giant, 900-kDa type I FAS (CpFAS1) that is capable of elongating medium-or long-chain fatty acids (42). On the other hand, Toxoplasma gondii and Eimeria tenella contain both type I and II enzymes and are able to synthesize fatty acids de novo and elongate long chain fatty acids (44). In addition, C. parvum also possesses a multifunctional polyketide synthase (CpPKS1) that is not present in any other apicompl-exans examined so far (43).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Functional Characterization of an Evolutionarily Distinct Phosphopantetheinyl Transferase in the Apicomplexan Cryptosporidium parvum

Cai¹,

Herschap²,

Zhu

2005

Eukaryot Cell

Self Cite

View full text Add to dashboard Cite

Recently, two types of fatty acid synthases (FASs) have been discovered from apicomplexan parasites. Although significant progress has been made in characterizing these apicomplexan FASs, virtually nothing was previously known about the activation and regulation of these enzymes. In this study, we report the discovery and characterization of two distinct types of phosphopantetheinyl transferase (PPTase) that are responsible for synthesizing holo-acyl carrier protein (ACP) from three apicomplexan parasites: surfactin production element (SFP) type in Cryptosporidium parvum (CpSFP-PPT), holo-ACP synthase (ACPS)-type in Plasmodium falciparum (PfACPS-PPT), and both SFP and ACPS types in Toxoplasma gondii (TgSFP-PPT and TgACPS-PPT). CpSFP-PPT and TgSFP-PPT are monofunctional, cytosolic, and phylogenetically related to animal PPTases. However, PfACPS-PPT and TgACPS-PPT are bifunctional (fused with a metal-dependent hydrolase), likely targeted to the apicoplast, and more closely related to proteobacterial PPTases. The function of apicomplexan PPTases has been confirmed by detailed functional analysis using recombinant CpSFP-PPT expressed from an artificially synthesized gene with codon usage optimized for Escherichia coli. The recombinant CpSFP-PPT was able to activate the ACP domains from the C. parvum type I FAS in vitro using either CoA or acetyl-CoA as a substrate, or in vivo when coexpressed in bacteria, with kinetic characteristics typical of PPTases. These observations suggest that the two types of fatty acid synthases in the Apicomplexa are activated and regulated by two evolutionarily distinct PPTases.

show abstract

“…C. parvum) or both (e.g. Toxoplasma gondii) (Roos et al, 2002;Zhu, 2004;Zhu et al, 2000bZhu et al, , 2002Zhu et al, , 2004. Although fatty acids are one of the major components in all cells, free fatty acids cannot enter any metabolic pathways unless they are activated by thioesterification with coenzyme A (CoA), to form an acyl-CoA ester, or with acyl carrier protein.…”

Section: Introductionmentioning

confidence: 99%

Functional characterization of a fatty acyl-CoA-binding protein (ACBP) from the apicomplexan Cryptosporidium parvum

2006

Self Cite

View full text Add to dashboard Cite

In this paper, the identification and functional analysis of a fatty acyl-CoA-binding protein (ACBP) gene from the opportunistic protist Cryptosporidium parvum are described. The CpACBP1 gene encodes a protein of 268 aa that is three times larger than typical ACBPs (i.e. ∼90 aa) of humans and animals. Sequence analysis indicated that the CpACBP1 protein consists of an N-terminal ACBP domain (∼90 aa) and a C-terminal ankyrin repeat sequence (∼170 aa). The entire CpACBP1 ORF was engineered into a maltose-binding protein fusion system and expressed as a recombinant protein for functional analysis. Acyl-CoA-binding assays clearly revealed that the preferred binding substrate for CpACBP1 is palmitoyl-CoA. RT-PCR, Western blotting and immunolabelling analyses clearly showed that the CpACBP1 gene is mainly expressed during the intracellular developmental stages and that the level increases during parasite development. Immunofluorescence microscopy showed that CpACBP1 is associated with the parasitophorous vacuole membrane (PVM), which implies that this protein may be involved in lipid remodelling in the PVM, or in the transport of fatty acids across the membrane.

show abstract

Expression and functional characterization of a giant Type I fatty acid synthase (CpFAS1) gene from Cryptosporidium parvum

Cited by 49 publications

References 33 publications

Functional characterization of the acyl-[acyl carrier protein] ligase in the Cryptosporidium parvum giant polyketide synthase

Functional characterization of the acyl-[acyl carrier protein] ligase in the Cryptosporidium parvum giant polyketide synthase

Functional Characterization of an Evolutionarily Distinct Phosphopantetheinyl Transferase in the Apicomplexan Cryptosporidium parvum

Functional characterization of a fatty acyl-CoA-binding protein (ACBP) from the apicomplexan Cryptosporidium parvum

Contact Info

Product

Resources

About