Functional Characterization of an Evolutionarily Distinct Phosphopantetheinyl Transferase in the Apicomplexan
            <i>Cryptosporidium parvum</i>

Cai, Xiwen; Herschap, Dustin; Zhu, Guangxi

doi:10.1128/ec.4.7.1211-1220.2005

Cited by 28 publications

(30 citation statements)

References 47 publications

(65 reference statements)

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“…The level of CpACBP1 transcripts was relatively low during early intracellular development (from 3 to 24 h post-infection), but gradually increased with the time of infection. This expression pattern differs from that of many other C. parvum genes, such as the replication protein A subunits, CpSFP-PPT and b-tubulin, but is similar to those of oocyst wall proteins in this parasite (Abrahamsen & Schroeder, 1999;Cai et al, 2005;Millership et al, 2004a;Rider et al, 2005). Such a differential expression pattern is also supported by Western blot and immunofluorescence microscopic analyses that only detected CpACBP1 protein in the intracellular parasites, but not in oocysts or free sporozoites (Figs 8 and 9).…”

Section: Cpacbp1 Has Highest Binding Affinity To C16 : 0 Palmitoyl-coasupporting

confidence: 53%

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

2006

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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.

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Section: Cpacbp1 Has Highest Binding Affinity To C16 : 0 Palmitoyl-coasupporting

confidence: 53%

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

2006

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“…It appears that all of these ACP domains need to be activated by the addition of a prosthetic phosphopantetheinyl moiety, which is catalyzed by PPT. Cryptosporidium possesses one single SFP-type PPT (CpSFP-PPT) that was able to activate the ACP domains in CpFAS1 (Cai et al, 2005). Here we have shown that CpSFP-PPT was able to activate the ACP domain in CpPKS1 (Fig.…”

Section: Discussionmentioning

confidence: 52%

“…Because recombinant ACP domains from CpFAS1 could only be expressed in the inactive apo form Cai et al, 2005), we suspected that the bacterial host cells were also unable to phosphopantetheinylate the ACP domain in the CpPKS1 loading unit. Therefore, we wanted to test the ability of recombinant C. parvum SFP-type phosphopantetheinyl transferase (CpSFP-PPT) to activate the apo-ACP domain of the CpPKS1 loading unit.…”

Section: Phosphopantetheinylation Of the Acp Domainmentioning

confidence: 99%

“…Therefore, we wanted to test the ability of recombinant C. parvum SFP-type phosphopantetheinyl transferase (CpSFP-PPT) to activate the apo-ACP domain of the CpPKS1 loading unit. CpSFP-PPT has been previously expressed as an S-tag-fused protein from an artificially synthesized DNA fragment, and its capacity in activating the ACP domains from CpFAS1 has been studied in detail (Cai et al, 2005). In this assay, a 40 μl reaction consisting of 75 mM Tris-HCl (pH 7.0), 10 mM MgCl 2 , 4 μg MBP-CpPKS1-AL-ACP, 400 ng CpSFP-PPT and 40 μM [1-14 C]acetylCoA was used so that the attachment of the phosphopantetheinyl group could be visualized by autoradiography.…”

Section: Phosphopantetheinylation Of the Acp Domainmentioning

confidence: 99%

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

Fritzler

Zhu

2007

International Journal for Parasitology

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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.

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“…To amplify C. parvum 18S rRNA as a control for normalization, we used the previously reported primers 995F (5Ј TAG AGA TTG GAG GTT GTT CCT 3Ј) and 1206R (5Ј CTC CAC CAA CTA AGA ACG GCC 3Ј) (1). The relative level of CpLCE1 transcripts was expressed relative to that of 18S rRNA, and values are reported based on at least three replicates as previously described (5,28).…”

Section: Methodsmentioning

confidence: 99%

Cryptosporidium parvum Long-Chain Fatty Acid Elongase

2007

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We report the presence of a new fatty acyl coenzyme A (acyl-CoA) elongation system in Cryptosporidium and the functional characterization of the key enzyme, a single long-chain fatty acid elongase (LCE), in this parasite. This enzyme contains conserved motifs and predicted transmembrane domains characteristic to the elongase family and is placed within the ELO6 family specific for saturated substrates. CpLCE1 gene transcripts are present at all life cycle stages, but the levels are highest in free sporozoites and in stages at 36 h and 60 h postinfection that typically contain free merozoites. Immunostaining revealed localization to the outer surface of sporozoites and to the parasitophorous vacuolar membrane. Recombinant CpLCE1 displayed allosteric kinetics towards malonyl-CoA and palmitoyl-CoA and Michaelis-Menten kinetics towards NADPH. Myristoyl-CoA (C 14:0 ) and palmitoyl-CoA (C 16:0 ) display the highest activity when used as substrates, and only one round of elongation occurs. CpLCE1 is fairly resistant to cerulenin, an inhibitor for both type I and II fatty acid synthases (i.e., maximum inhibitions of 20.5% and 32.7% were observed when C 16:0 and C 14:0 were used as substrates, respectively). These observations ultimately validate the function of CpLCE1.As one of the vital compounds for all organisms, fatty acids of 14 to 18 carbons in length comprise the bulk of cellular fatty acids that serve structural and biological functions, and they are usually the major products of de novo synthesis in most cells. These long-chain fatty acids play important roles in many biological functions such as energy metabolism and membrane structure. There are several metabolic pathways that produce long-chain fatty acids, most notably the type I and type II fatty acid synthases (FASs). The type I enzymes of mammals and fungi are typically cytosolic and composed of multiple enzymes arranged into domains of one or two large polypeptides (31). In contrast, the enzymes of the type II FASs are all located on separate domains and are found in prokaryotes or in eukaryotic organelles of prokaryotic origin (33).Relatively common among eukaryotic organisms are the fatty acid elongase-based systems. These elongase-based systems directly elongate a fatty acyl chain esterfied with CoA (fatty acyl-CoA), which is in contrast to the type I and type II FAS systems, which elongate a fatty acyl chain attached to an acyl carrier protein. The elongase system is comprised of at least four enzymes that are responsible for adding two carbon units to the fatty acyl carboxyl end. The chemistry of this pathway is similar to that used by type I and type II FASs. Fatty acyl elongation begins with the condensation of malonyl-CoA with a fatty acyl-CoA catalyzed by the condensing enzyme LCE (␤-ketoacyl-CoA synthase) (Fig.

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

Cited by 28 publications

References 47 publications

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

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

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

Cryptosporidium parvum Long-Chain Fatty Acid Elongase

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