Interactions of Streptomyces griseus aminopeptidase with a methionine product analogue:: a structural study at 1.53 Å resolution
Interactions of Streptomyces griseus aminopeptidase with a methionine product analogue:: a structural study at 1.53 Å resolution
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DOI：
10.1107/s0907444900002420
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发表时间：
2000-05-01
期刊：
影响因子：
--
通讯作者：
Shoham, G
中科院分区：
文献类型：
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作者：
Gilboa, R;Greenblatt, HM;Shoham, G
SGAP是一种存在于灰色链霉菌细胞外液中的氨基肽酶。它是一种双锌酶，对大的疏水氨基末端残基有很强的偏好。它是一种单体（30kda）热稳定酶，具有钙离子调节的高效催化活性。SGAP具有体积小、活性高、热稳定性好等特点，是一种具有广泛应用前景的酶。迄今为止，仅有一种相关的氨肽酶（Aeromonas proteolytica AP； AAP）进行了结构分析，结果显示其结构与SGAP非常相似，尽管这两种酶之间的序列同源性较低。对SGAP进行详细结构分析的动机源于对双锌氨基肽酶家族的强烈机制兴趣，以及这些酶的高潜在适用性。原生SGAP的1.75埃晶体结构先前已被报道，但由于活性位点周围的结构区域不确定，因此不允许进行关键的机制解释。本文报道了SGAP在1.58埃分辨率下更为精确的结构，以及SGAP与抑制型甲硫氨酸配合物的1.53埃分辨率结构，后者也是SGAP催化过程的产物。这两种高分辨率结构可以更好地理解底物和产物的SGAP结合模式。这些研究允许追踪酶的先前紊乱区域（Glu196-Arg202），并鉴定酶的一些参与酶-底物相互作用的功能基团（Asp160, Met161, Gly201， Arg202和Phe219）。这些研究还表明，Glu131可能作为水解亲核试剂直接参与SGAP的催化机制。结构结果与最近的AAP结构进行了比较，以得出与该低分子量氨基肽酶家族相关的一般功能结论。
SGAP is an aminopeptidase present in the extracellular fluid of Streptomyces griseus cultures. It is a double-zinc enzyme with a strong preference for large hydrophobic amino-terminus residues. It is a monomeric (30 kDa) heat-stable enzyme, with a high and efficient catalytic activity modulated by calcium ions. The small size, high activity and heat stability make SGAP a very attractive enzyme for various biotechnological applications. Only one other related aminopeptidase (Aeromonas proteolytica AP; AAP) has been structurally analyzed to date and its structure was shown to be considerably similar to SGAP, despite the low sequence homology between the two enzymes. The motivation for the detailed structural analysis of SGAP originated from a strong mechanistic interest in the family of double-zinc aminopeptidases, combined with the high potential applicability of these enzymes. The 1.75 Angstrom crystallographic structure of native SGAP has been previously reported, but did not allow critical mechanistic interpretations owing to inconclusive structural regions around the active site. A more accurate structure of SGAP at 1.58 Angstrom resolution is reported in this paper, along with the 1.53 Angstrom resolution structure of the SGAP complex with inhibitory methionine, which is also a product of the SGAP catalytic process. These two high-resolution structures enable a better understanding of the SGAP binding mode of both substrates and products. These studies allowed the tracing of the previously disordered region of the enzyme (Glu196-Arg202) and the identification of some of the functional groups of the enzyme that are involved in enzyme-substrate interactions (Asp160, Met161, Gly201, Arg202 and Phe219). These studies also suggest that Glu131 is directly involved in the catalytic mechanism of SGAP, probably as the hydrolytic nucleophile. The structural results are compared with a recent structure of AAP with an hydroxamate inhibitor in order to draw general functional conclusions which are relevant for this family of low molecular-weight aminopeptidases.