Persson K, Aslund L, Grahn B, Hanke J, Heby O
Department of Cellular and Developmental Biology, Umeâ University, S-901 87 Umeå, Sweden.
Biochem J. 1998 Aug 1;333 ( Pt 3)(Pt 3):527-37. doi: 10.1042/bj3330527.
All attempts to identify ornithine decarboxylase in the human pathogen Trypanosoma cruzi have failed. The parasites have instead been assumed to depend on putrescine uptake and S-adenosylmethionine decarboxylase (AdoMetDC) for their synthesis of the polyamines spermidine and spermine. We have now identified the gene encoding AdoMetDC in T. cruzi by PCR cloning, with degenerate primers corresponding to conserved amino acid sequences in AdoMetDC proteins of other trypanosomatids. The amplified DNA fragment was used as a probe to isolate the complete AdoMetDC gene from a T. cruzi genomic library. The AdoMetDC gene was located on chromosomes with a size of approx. 1.4 Mbp, and contained a coding region of 1110 bp, specifying a sequence of 370 amino acid residues. The protein showed a sequence identity of only 25% with human AdoMetDC, the major differences being additional amino acids present in the terminal regions of the T. cruzi enzyme. As expected, a higher sequence identity (68-72%) was found in comparison with trypanosomatid AdoMetDCs. When the coding region was expressed in Escherichia coli, the recombinant protein underwent autocatalytic cleavage, generating a 33-34 kDa alpha subunit and a 9 kDa beta subunit. The encoded protein catalysed the decarboxylation of AdoMet (Km 0.21 mM) and was stimulated by putrescine but inhibited by the polyamines, weakly by spermidine and strongly by spermine. Methylglyoxal-bis(guanylhydrazone) (MGBG), a potent inhibitor of human AdoMetDC, was a poor inhibitor of the T. cruzi enzyme. This differential sensitivity to MGBG suggests that the two enzymes are sufficiently different to warrant the search for compounds that might interfere with the progression of Chagas' disease by selectively inhibiting T. cruzi AdoMetDC.
在人类病原体克氏锥虫中鉴定鸟氨酸脱羧酶的所有尝试均告失败。相反，人们认为这些寄生虫依赖于腐胺摄取和S-腺苷甲硫氨酸脱羧酶（AdoMetDC）来合成多胺亚精胺和精胺。我们现在通过PCR克隆，使用与其他锥虫的AdoMetDC蛋白中保守氨基酸序列相对应的简并引物，在克氏锥虫中鉴定了编码AdoMetDC的基因。扩增的DNA片段用作探针，从克氏锥虫基因组文库中分离出完整的AdoMetDC基因。AdoMetDC基因位于大小约为1.4 Mbp的染色体上，包含一个1110 bp的编码区，指定了一个370个氨基酸残基的序列。该蛋白与人类AdoMetDC的序列同一性仅为25%，主要差异在于克氏锥虫酶末端区域存在额外的氨基酸。正如预期的那样，与锥虫的AdoMetDC相比，发现了更高的序列同一性（68 - 72%）。当编码区在大肠杆菌中表达时，重组蛋白进行自催化切割，产生一个33 - 34 kDa的α亚基和一个9 kDa的β亚基。编码的蛋白催化AdoMet的脱羧反应（Km为0.21 mM），受腐胺刺激，但受多胺抑制，受亚精胺的抑制作用较弱，受精胺的抑制作用较强。甲基乙二醛双（胍腙）（MGBG）是人类AdoMetDC的有效抑制剂，对克氏锥虫酶的抑制作用较弱。这种对MGBG的差异敏感性表明，这两种酶有足够的差异，值得寻找可能通过选择性抑制克氏锥虫AdoMetDC来干扰恰加斯病进展的化合物。
