Two beta-alanyl-CoA:ammonia lyases in Clostridium propionicum
Two beta-alanyl-CoA:ammonia lyases in Clostridium propionicum
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DOI：
10.1111/j.1742-4658.2004.04518.x
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发表时间：
2005-02-01
期刊：
影响因子：
5.4
通讯作者：
Buckel, W
中科院分区：
文献类型：
--
作者：
Herrmann, G;Selmer, T;Buckel, W
丙酸梭菌发酵 P-丙氨酸首先活化为 CoA-硫羟酸酯，然后脱氨基为丙烯酰-CoA，丙烯酰-CoA 也是 L-丙氨酸发酵的中间体。通过将生物体从碳和能源 α-丙氨酸转变为 P-丙氨酸，β-丙氨酰辅酶A：氨裂合酶被诱导 300 倍（约可溶性蛋白质的 30%）。低基础裂解酶活性由 acl1 基因编码，而几乎相同的 acl2 基因（六个氨基酸取代）负责在 β-丙氨酸上生长后的高活性。推导的 β-丙氨酰-CoA：氨裂解酶蛋白与参与赖氨酸发酵的推定 β-氨基丁酰-CoA 氨裂解酶有关，并在几种厌氧细菌的基因组中发现。 β-丙氨酰辅酶A：氨裂解酶 2 被纯化至均质，并被表征为由 16 kDa 亚基组成的异五聚体。测量丙烯酰辅酶A的表观K-m值为23+/-4μM，与第二底物氨的浓度无关； k(cat)/K-m 计算为 10(7) M-1 s(-1)。氨的表观 K-m 更高，在 150 muM 丙烯酰辅酶 A 下为 70 +/- 5 mM，k(cat)/K-m 低得多，为 4 x 10(3) M-1.s(-1)。在逆反应中，β-丙氨酰-CoA 的 K-m 为 210 +/- 30 muM。 100 mm 氯化铵对氨的消除有 70% 的抑制。 β-丙氨酸生长的细胞中β-丙氨酰辅酶A：氨裂解酶的含量比维持生物体生长速度所需的含量高约100倍。因此，建议需要该酶来结合丙烯酰辅酶A，以将有毒游离形式保持在非常低的水平。推导了L-丙氨酸/β-丙氨酸或D-乳酸/3-羟基丙酸之间异构化平衡的计算公式。
The fermentation of P-alanine by Clostridium propionicum proceeds via activation to the CoA-thiol ester, followed by deamination to acryloyl-CoA, which is also an intermediate in the fermentation Of L-alanine. By shifting the organism from the carbon and energy source alpha-alanine to P-alanine, the enzyme beta-alanyl-CoA:ammonia lyase is induced 300-fold (approximate to 30% of the soluble protein). The low basal lyase activity is encoded by the acl1 gene, whereas the almost identical acl2 gene (six amino acid substitutions) is responsible for the high activity after growth on beta-alanine. The deduced beta-alanyl-CoA:ammonia lyase proteins are related to putative beta-aminobutyryl-CoA ammonia lyases involved in lysine fermentation and found in the genomes of several anaerobic bacteria. beta-Alanyl-CoA:ammonia lyase 2 was purified to homogeneity and characterized as a heteropentamer composed of 16 kDa subunits. The apparent K-m value for acryloyl-CoA was measured as 23 +/- 4 muM, independent of the concentration of the second substrate ammonia; k(cat)/K-m was calculated as 10(7) M-1 s(-1). The apparent K-m for ammonia was much higher, 70 +/- 5 mM at 150 muM acryloyl-CoA with a much lower k(cat)/K-m of 4 x 10(3) M-1.s(-1). In the reverse reaction, a K-m of 210 +/- 30 muM was obtained for beta-alanyl-CoA. The elimination of ammonia was inhibited by 70% at 100 mm ammonium chloride. The content of beta-alanyl-CoA:ammonia lyase in beta-alanine grown cells is about 100 times higher than that required to sustain the growth rate of the organism. It is therefore suggested that the enzyme is needed to bind acryloyl-CoA, in order to keep the toxic free form at a very low level. A formula was derived for the calculation of isomerization equilibra between L-alanine/beta-alanine or D-lactate/3-hydroxypropionate.