Department of Veterinary Pathobiology, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, Texas 77843-4467, USA.
BMC Biochem. 2010 Nov 22;11:46. doi: 10.1186/1471-2091-11-46.
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.
顶复门生物微小隐孢子虫基因组拥有一个 25kb 无内含子的开放阅读框（ORF），预测其编码的多功能 I 型脂肪酸合酶（CpFAS1）具有至少 21 个酶结构域。虽然 CpFAS1 的结构类似于细菌聚酮合酶（PKS），但这种巨合酶被预测为脂肪酸酰基延长酶，因为我们之前的研究表明，N 端加载单元（酰基-[ACP]连接酶）更喜欢使用中链到长链脂肪酸作为底物，并且三个内部延伸模块中的每一个都包含一套完整的酶来产生饱和脂肪酸酰基链。尽管使用重组蛋白已经证实了几乎所有结构域的活性，但 C 端还原酶结构域（CpFAS1-R）的活性尚未确定。事实上，没有发表的研究使用纯化的重组或天然蛋白来报道细菌 PKS 中任何还原酶结构域的动力学特征。
通过序列相似性和特征基序的计算机分析，本研究证实了 CpFAS1-R 作为还原酶的身份。基于 R 结构域的系统发育分析支持了顶复门生物 I 型 FAS/PKS 基因的细菌起源的先前观点。我们还开发了一种使用脂肪酸酰基辅酶 A 作为底物的新测定方法，并确定 CpFAS1-R 只能利用非常长链脂肪酸酰基辅酶 A 作为底物（即对 C26 > C24 > C22 > C20 有活性，但对 C18 和 C16 没有活性）。它能够使用 NADPH 和 NADH 作为电子供体，但更喜欢 NADPH 而不是 NADH。CpFAS1-R 对 C26 十六烷酰基辅酶 A 作为底物的活性表现出变构动力学（h = 2.0；Vmax = 32.8 nmol min-1 mg-1 蛋白；K50 = 0.91 mM）。
我们通过直接测定其底物偏好和动力学参数来证实了 CpFAS1-R 的活性，这是首次对 I 型 FAS、PKS 或非核糖体肽合酶（NRPS）还原酶结构域进行的测定。对非常长链脂肪酸硫酯的受限底物偏好可能是该巨合酶避免释放不期望长度的产物的重要特征。
