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首页> 外文期刊>Journal of Molecular Biology >High-resolution crystal structure of Arthrobacter aurescens chondroitin AC lyase: an enzyme-substrate complex defines the catalytic mechanism.
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High-resolution crystal structure of Arthrobacter aurescens chondroitin AC lyase: an enzyme-substrate complex defines the catalytic mechanism.

机译：金色节杆菌软骨素AC裂解酶的高分辨率晶体结构：酶-底物复合物定义了催化机理。

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摘要

Chondroitin lyases (EC 4.2.2.4 and EC 4.2.2.5) are glycosaminoglycan-degrading enzymes that act as eliminases. Chondroitin lyase AC from Arthrobacter aurescens (ArthroAC) is known to act on chondroitin 4-sulfate and chondroitin 6-sulfate but not on dermatan sulfate. Like other chondroitin AC lyases, it is capable of cleaving hyaluronan. We have determined the three-dimensional crystal structure of ArthroAC in its native form as well as in complex with its substrates (chondroitin 4-sulfate tetrasaccharide, CS(tetra) and hyaluronan tetrasaccharide) at resolution varying from 1.25 A to 1.9A. The primary sequence of ArthroAC has not been previously determined but it was possible to determine the amino acid sequence of this enzyme from the high-resolution electron density maps and to confirm it by mass spectrometry. The enzyme-substrate complexes were obtained by soaking the substrate into the crystals for varying lengths of time (30 seconds to ten hours) and flash-cooling the crystals. The electron densitymap for crystals soaked in the substrate for as short as 30 seconds showed the substrate clearly and indicated that the ring of central glucuronic acid assumes a distorted boat conformation. This structure strongly supports the lytic mechanism where Tyr242 acts as a general base that abstracts the proton from the C5 position of glucuronic acid while Asn183 and His233 neutralize the charge on the glucuronate acidic group. Comparison of this structure with that of chondroitinase AC from Flavobacterium heparinum (FlavoAC) provides an explanation for the exolytic and endolytic mode of action of ArthroAC and FlavoAC, respectively.
机译：软骨素裂解酶（EC 4.2.2.4和EC 4.2.2.5）是糖胺聚糖降解酶，可作为消除酶。已知来自金色节杆菌（ArthroAC）的软骨素裂合酶AC作用于4-硫酸软骨素和6-硫酸软骨素，但不作用于硫酸皮肤素。像其他软骨素AC裂解酶一样，它能够裂解透明质酸。我们已经确定了ArthroAC的三维晶体结构，其天然形式以及与其底物（软骨素4-硫酸盐四糖，CS（四）和透明质酸四糖）的复合物的分辨率在1.25 A至1.9A之间变化。此前尚未确定ArthroAC的主要序列，但可以从高分辨率电子密度图确定该酶的氨基酸序列，并通过质谱法对其进行确认。通过将底物浸入晶体中不同的时间长度（30秒至十小时）并快速冷却晶体，可以得到酶-底物复合物。浸泡在基质中短至30秒的晶体的电子密度图清楚地显示了基质，并表明中央葡糖醛酸的环呈扭曲的船形。这种结构有力地支持了裂解机制，其中Tyr242充当了将质子从葡萄糖醛酸的C5位置提取出来的一般碱基，而Asn183和His233中和了葡萄糖醛酸酸性基团上的电荷。将该结构与来自肝黄杆菌的软骨素酶AC（FlavoAC）的结构进行比较，分别解释了ArthroAC和FlavoAC的外切和内切作用方式。

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