Purification and initial characterization of proline 4-hydroxylase from Streptomyces griseoviridus P8648: A 2-oxoacid, ferrous-dependent dioxygenase involved in etamycin biosynthesis
Purification and initial characterization of proline 4-hydroxylase from Streptomyces griseoviridus P8648: A 2-oxoacid, ferrous-dependent dioxygenase involved in etamycin biosynthesis
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DOI：
10.1042/bj3130185
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发表时间：
1996-01-01
影响因子：
4.1
通讯作者：
Schofield, CJ
中科院分区：
文献类型：
--
作者：
Lawrence, CC;Sobey, WJ;Schofield, CJ
脯氨酸4-羟化酶是一种2-氧酸、铁离子依赖的双加氧酶，参与次级代谢物埃他霉素的生物合成。本文报道了从灰状病毒链霉菌P8648中低产量纯化脯氨酸4-羟化酶并对其进行初步鉴定。在大多数情况下，脯氨酸4-羟化酶是2-氧酸依赖性双加氧酶家族的典型成员。它是单体(M(r)近似。38000)（通过Superdex-G75凝胶过滤），通常对亚铁离子和2-氧葡萄糖酸盐有严格的要求。该酶被2-氧戊二酸的芳香类似物抑制。观察到l -脯氨酸不偶联的2-氧葡萄糖酸酯转化为琥珀酸和CO2。l -抗坏血酸的添加不刺激l -脯氨酸偶联的2-氧戊二酸的转换，但刺激l -脯氨酸不偶联的转换。l -抗坏血酸对l -脯氨酸羟基化有时间依赖性的抑制作用，在组氨酸修饰条件下，用焦碳酸二乙酯预孵育可使该酶完全失活。这种失活可以通过在预孵育混合物中包含l -脯氨酸和2-氧戊二酸来部分阻止，这表明在活性位点存在组氨酸残基。
Proline 4-hydroxylase is a 2-oxoacid, ferrous-ion-dependent dioxygenase involved in the biosynthesis of the secondary metabolite etamycin. The purification, in low yield, of proline 4-hydroxylase from Streptomyces griseoviridus P8648 to near apparent homogeneity and its initial characterization are reported. In most respects proline 4-hydroxylase is a typical member of the 2-oxoacid-dependent dioxygenase family. It is monomeric (M(r) approx. 38000) (by gel filtration on Superdex-G75) and has typically strict requirements for ferrous ion and 2-oxoglutarate. The enzyme was inhibited by aromatic analogues of 2-oxoglutarate. L-Proline-uncoupled turnover of 2-oxoglutarate to succinate and CO2 was observed. The addition of L-ascorbate did not stimulate L-proline-coupled turnover of 2-oxoglutarate, but did stimulate L-proline-uncoupled turnover. L-Ascorbate caused a time-dependent inhibition of L-proline hydroxylation, The enzyme was completely inactivated by preincubation with diethyl pyrocarbonate under histidine-modifying conditions. This inactivation could be partially prevented by the inclusion of L-proline and 2-oxoglutarate in the preincubation mixture, suggesting the presence of histidine residue(s) at the active site.