陈晓迪1,3, 王凤山2,3, 肖敏1,2
![通讯作者](http://journals.im.ac.cn/html/actamicrocn/2017/8/images/REcor.gif)
![minxiao@sdu.edu.cn](http://journals.im.ac.cn/html/actamicrocn/2017/8/images/REemail.gif)
1.山东大学微生物技术国家重点实验室, 山东 济南 250100;
2.国家糖工程技术研究中心, 山东省糖化学生物学省级重点实验室, 山东 济南 250100;
3.山东大学药学院, 山东 济南 250100
收稿日期:2017-03-30;修回日期:2017-05-21;网络出版日期:2017-05-25
基金项目:国家重点基础研究发展计划(国家"973计划")(2012CB822102);国家自然科学基金(31670062)
*通信作者:肖敏, Tel:+86-531-88365128;Fax:+86-531-88363002;E-mail:minxiao@sdu.edu.cn
摘要:β-N-乙酰氨基己糖苷酶(EC.3.2.1.52)是一类重要的糖苷水解酶,在自然界中催化简单的β-N-乙酰氨基己糖苷或复杂的寡糖链、多糖链中末端N-乙酰己糖苷键的水解,在微生物、植物和动物中广泛分布,具有重要的生物学功能。某些种类的β-N-乙酰氨基己糖苷酶在一定的人为条件下水解β-N-乙酰氨基己糖苷键的同时还具有转糖基作用,能将β-N-乙酰氨基己糖基转移到不同的羟基化合物上,合成β-N-乙酰氨基己糖苷化合物,在糖链合成上具有应用的潜力。本文综述了β-N-乙酰氨基己糖苷酶的结构和催化机制、酶的生物学功能以及酶在β-N-乙酰氨基己糖苷化合物合成中的应用,以促进β-N-乙酰氨基己糖苷酶的进一步研究和开发应用。
关键词: β-N-乙酰氨基己糖苷酶 催化机制 生物学功能 转糖基作用 寡糖合成
β-N-Acetylhexosaminidases and their application in the synthesis of β-N-acetyl-D-hexosaminides
Xiaodi Chen1,3, Fengshan Wang2,3, Min Xiao1,2
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1.State Key Laboratory of Microbial Technology, Jinan 250100, Shandong Province, China;
2.National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, Jinan 250100, Shandong Province, China;
3.School of Pharmaceutical Sciences, Shandong University, Jinan 250100, Shandong Province, China
Received 30 March 2017; Revised 21 May 2017; Published online 25 May 2017
*Corresponding author: Min Xiao, Tel:+86-531-88365128;Fax:+86-531-88363002;E-mail:minxiao@sdu.edu.cn
Supported by Major State Basic Research Development Program of China (2012CB822102) and by the National NaturalScience Foundation of China (31670062)
Abstract: β-N-Acetylhexosaminidases (EC.3.2.1.52) are an important class of glycosidases that catalyze hydrolysis of terminal N-acetyl-β-D-hexosamine from various oligosaccharides and polysaccharides. These enzymes are widely distributed in microorganisms, plants, and animals, and play crucial roles in nature. Some β-N-acetylhexosaminidases can catalyze glycosyl transfer to form β-N-acetyl-D-hexosaminides, which have shown great potentiality in enzymatic synthesis of functional glycans. In this review, catalytic mechanisms and biological functions of β-N-acetylhexosaminidases, and potential applications of these enzymes in the synthesis of β-N-acetyl-D-hexosaminide are summarized.
Key words: β-N-Acetylhexosaminidase catalytic mechanism biological function transglycosylation oligosaccharide synthesis
β-N-乙酰氨基己糖苷酶(β-N-acetylhexosaminidase,EC.3.2.1.52) 是一类重要的糖苷水解酶,在自然界中催化简单的β-N-乙酰氨基己糖苷或复杂的寡糖链、多糖链中末端N-乙酰半乳糖苷键或N-乙酰葡萄糖苷键的水解。20世纪70年代,Matsushima等首先从高峰淀粉酶(Takadiastase)中分离出了β-N-乙酰氨基己糖苷酶,并在水解底物特异性和抑制剂等方面进行了系统研究,揭示了该类酶的催化特性[1]。近年来,随着对β-N-乙酰氨基己糖苷酶的深入研究,人们发现了某些种类的β-N-乙酰氨基己糖苷酶在一定的人为条件下水解β-N-乙酰氨基己糖苷键的同时,还可以通过转糖基或逆水解作用将β-N-乙酰氨基己糖基转移到不同的羟基化合物上,合成β-N-乙酰氨基己糖苷化合物,用于合成多种功能性寡糖,具有重要的应用前景[2]。本文综述了β-N-乙酰氨基己糖苷酶的结构和催化机制、酶的生物学功能以及酶在β-N-乙酰氨基己糖苷化合物合成中的应用研究进展。
1 β-N-乙酰氨基己糖苷酶的结构和催化机制 通过对CAZY数据库(http://www.cazy.org/Glycoside-Hydrolases.html)分析,目前β-N-乙酰氨基己糖苷酶(EC.3.2.1.52) 有141种,主要分布在糖苷酶家族GH3、GH20和GH84中,而在GH116家族仅有一例β-N-乙酰氨基葡萄糖苷酶的报道[3]。大量的β-N-乙酰氨基己糖苷酶的晶体结构研究结果显示,该类酶的催化结构域均为(β/α)8桶状结构,由8个重复的β折叠/loop环/α螺旋单位组成,8个β折叠组成了催化域的β桶。β-N-乙酰氨基己糖苷酶有两种不同的催化机制,一是糖苷酶通用的构型保持酶的双置换机制,即首先通过亲核体氨基酸Asp/Glu直接对底物进行攻击从而形成酶-底物复合物中间体,酸碱催化氨基酸Asp-His/Asp先提供质子,促使离去基团离去,然后进行碱催化,激活受体分子,促使受体分子攻击酶-底物复合物,当受体为水时发生水解反应,当受体为糖时发生转糖基反应(图 1-A和B)[3-4]。二是底物辅助型的催化机制,发挥亲核作用的是底物分子中的HexNAc而非氨基酸,即HexNAc的2位乙酰氨基极化并被去质子化的Asp辅助放置于利于酸碱催化氨基酸Asp/Glu进行反应的位置,乙酰氨基对异头碳进行亲核攻击形成恶唑啉中间体,Asp/Glu提供质子,促使离去基团离去,随后进行碱催化,激活受体分子,促使受体分子攻击中间体,受体为水时发生水解反应,受体为糖时发生转糖基反应(图 1-C)[2]。迄今为止,多种来自GH3、GH20和GH84三个家族的β-N-乙酰氨基己糖苷酶的晶体结构已经得到解析,而仅有一例β-N-乙酰氨基葡萄糖苷酶的GH116家族的酶尚未发现晶体结构解析的报道。
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图 1 β-N-乙酰氨基己糖苷酶的催化机制 Figure 1 Catalytic mechanisms for β-N-acetylhexosaminidases. A: Putative double-displacement retainingmechanism utilized by GH116. B: Double-displacement retaining mechanism utilized by GH3. A histidine isproposed to act as the general acid/base. C: Double-displacement retaining mechanism of GH20 and GH84. Thenucleophile is not provided by the enzyme but by the substrate 2-acetamido group, leading to the formation of anoxazoline intermediate. |
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1.1 GH3家族β-N-乙酰氨基己糖苷酶 GH3家族中归于β-N-乙酰氨基己糖苷酶的有18种来源于细菌[4-8],仅1种来源于真菌[9],该家族的β-N-乙酰氨基己糖苷酶专一性水解β-N-乙酰氨基葡萄糖苷键,为β-N-乙酰氨基葡萄糖苷酶。第1种得到蛋白质晶体结构解析的是2007年报道的霍乱弧菌(Vibrio cholerae) β-N-乙酰氨基己糖苷酶VcNagZ[5],之后又有4种细菌源的β-N-乙酰氨基己糖苷酶晶体结构陆续得到解析。2015年,Qin等报道了至今该家族中仅有的一种真菌米黑根毛霉(Rhizomucor miehei)来源的β-N-乙酰氨基己糖苷酶的晶体结构[9](表 1)。2010年,Litzinger等对来自枯草杆菌的β-N-乙酰氨基葡萄糖苷酶BsNagZ的晶体以及BsNagZ与过渡态模拟抑制剂PUGNAc的共晶体进行了结构解析(表 1),是第一个报道的具有双结构域的β-N-乙酰氨基己糖苷酶,其N端结构域为典型的糖苷酶(β/α)8桶状结构催化结构域,亲核体氨基酸Asp和酸碱催化对Asp-His相距约6.3?,分别位于第5和第7个β折叠的末端(图 2-B)[4];C端结构域为αβα三明治结构,与底物或抑制物的结合位点无关。该报道首次阐明了GH3家族的β-N-乙酰氨基葡萄糖苷酶催化机制为双置换反应机制(图 1-B)[4],与糖苷酶一般以Glu作为酸碱催化氨基酸不同的是该家族以Asp-His作为酸碱催化对,其中His主要行使酸碱催化功能,其与Asp形成氢键,组成了Asp-His催化对(图 2-A和B)。与枯草杆菌的β-N-乙酰氨基葡萄糖苷酶BsNagZ不同的是,霍乱弧菌(Vibriocholerae)和沙门氏菌(Salmonella typhimurium)的β-N-乙酰氨基葡萄糖苷酶VcNagZ和StNagZ是单结构域,即只有1个(β/α)8桶状的催化结构;而2个来自热袍菌属的海栖热袍菌(Thermotogamaritime)和新阿波罗栖热袍菌(Thermotoganeapolitana)的β-N-乙酰氨基葡萄糖苷酶NagA和CbsA,具有N端催化结构域和C端结构域双结构域酶,但与已报道的该家族细菌源单结构域和双结构域β-N-乙酰氨基葡萄糖苷酶晶体结构均为单体蛋白不同的是,NagA和CbsA为二聚体蛋白。GH3家族仅有的一种真菌米黑根毛霉(R. miehei)RmNag的晶体结构与细菌源β-N-乙酰氨基葡萄糖苷酶有很大不同,是一个双功能酶,由4个不同的结构域组成,分别为N端的A和B结构域以及C端的C和D结构域。N端为双结构域β-N-乙酰氨基葡萄糖苷酶,其中A结构域为(β/α)8桶状的催化结构,B结构域为3α/6β/3α的三明治结构;C端为双结构域的乙酰转移酶,包含2个结构上保守的GCN5相关乙酰转移酶结构域C和D。
表 1. β-N-乙酰氨基己糖苷酶的晶体结构 Table 1. Three dimensional structures of β-N-acetylhexosaminidases
GH family | Enzyme | PDB | Carbohydrate ligands | Enzyme source | Ref |
3 | BsNagZ | 3BMX | Bacillus subtilis | [4] | |
3NVD | PUGNAc | ||||
N318D | 3LK6 | ||||
N318D | 4GYJ | GlcNAc-MurNAc | |||
N318D | 4GYK | GlcNAc-MurNAc | |||
3 | StNagZ | 4GVF | GlcNAc | Salmonella | [6] |
4GVG | typhimurium | ||||
4GVH | 5-fluoro-GlcNAc | ||||
4GVI | GlcNAc-1, 6-anhMurNAc | ||||
4HVM | C1 | ||||
3 | NagA | 3WO8 | Thermotoga maritima | [7] | |
3 | CbsA | 5BZA | Thermotoga | [8] | |
5C0Q | neapolitana | ||||
3 | VcNagZ | 1TR9 | Vibrio cholerae | [5] | |
1Y65 | GlcNAc | ||||
2OXN | PUGNAc | ||||
3GS6 | N-butyryl-PUGNAc | ||||
3GSM | N-Valeryl-PUGNAc | ||||
3 | RmNag | 4ZM6 | Rhizomucor miehei | [9] | |
20 | AAur_4089 | 3RCN | Paenarthrobacter aurescens | unpublished | |
20 | Hex1-ΔC | 3GH4 | Paenibacillus sp. | [10] | |
3GH5 | GlcNAc | ||||
3GH7 | GalNAc | ||||
3SUR | NAG-thiazoline | ||||
3SUS | GalNAG-thiazoline | ||||
3SUT | PUGNAc | ||||
3SUU | GalPUGNAc | ||||
3SUV | NHAc-DNJ | ||||
3SUW | NHAc-CAS | ||||
20 | SmGH20A | 1QBA | Serratia marcescens | [11] | |
1QBB | di-N-acetyl-β-D-glucosamine | ||||
D539A | 1C7S | di-N-acetyl-β-D-glucosamine | |||
E540D | 1C7T | di-N-acetyl-β-D-glucosamine | |||
20 | GcnA | 2EPK | Streptococcus gordonii | [12] | |
2EPL | |||||
2EPM | |||||
2EPN | |||||
2EPN | |||||
20 | StrH | 3RPM | GlcNAc | Streptococcus | [13] |
pneumoniae | |||||
20 | GH20C | 5A69 | Gal-PUGNAc | Streptococcus | [14] |
5A6A | NGT | pneumoniae | |||
5A6B | PUGNAc | ||||
5A6K | Gal-NGT | ||||
5AC4 | GalNAc | ||||
5AC5 | GlcNAc | ||||
20 | StrH | 2YL6 | Streptococcus | [15] | |
2YL8 | GlcNAc-Man | pneumoniae | |||
2YLL | |||||
4AZ5 | |||||
4AZ6 | PUGNAc | ||||
4AZ7 | LOGNAC | ||||
4AZB | PUGNAc | ||||
2YL5 | |||||
2YL9 | GlcNAc-Man-Man-GlcNAc | ||||
2YLA | GlcNAc-Man-Man-GlcNAc | ||||
4AZC | |||||
4AZG | PUGNAc | ||||
4AZH | LOGNAC | ||||
4AZI | PUGNAc | ||||
20 | ScGH20 | 4C7D | Streptomyces | [16] | |
4C7F | 6-acetamido-6-deoxy-castanospermine | coelicolor | |||
4C7G | GlcNAc-oxazoliniumion | ||||
20 | SpHex | 1HP4 | Streptomyces plicatus | [17-18] | |
1HP5 | NAG-thiazoline | ||||
1JAK | C2 | ||||
1M01 | GlcNAc | ||||
D313A | 1M03 | GlcNAc | |||
D313N | 1M04 | GlcNAc | |||
5FCZ | Thio-NAglucal | ||||
5FD0 | NAGlucal | ||||
20 | HexA | 2GJX | Homo sapiens | [19] | |
2GK1 | NAG-thiazoline | ||||
HexB | 1NOU | Homo sapiens | [20] | ||
1NOW | GalNAc-isofagomine | ||||
1NP0 | NAG-thiazoline | ||||
1O7A | |||||
3LMY | Pyrimethamine | ||||
5BRO | |||||
20 | OfHex1 | 3NSM | Ostrinia furnacalis | [21] | |
3NSN | TMG-chitotriomycin | ||||
3OZO | NAG-thiazoline | ||||
3OZP | PUGNAc | ||||
V327G | 3S6T | PUGNAc | |||
3WMB | naphthalimide derivative Q1 | ||||
3WMC | naphthalimide derivative Q2 | ||||
84 | CpNagJ | 2J62 | GlcNAcstatin | Clostridium | [22] |
2V5C | |||||
2V5D | |||||
2VUR | C3 | ||||
2WB5 | GlcNAcstatin | ||||
2X0Y | Diprophylline | ||||
D298N | 2YDQ | hOGA-derived O-GlcNAc peptide | |||
D298N | 2YDR | p53-derived O-GlcNAc peptide | |||
D298N | 2YDS | TAB 1-derived O-GlcNAc peptide | |||
D298N | 4ZXL | C4 | |||
84 | NagJ | 2CBI | DIHYDROFURAN-2(3H)-ONE | Clostridium | [23] |
2CBJ | PUGNAC | perfringens | |||
2XPK | GlcNAcstatin F |
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图 2 β-N-乙酰氨基己糖苷酶的催化位点及催化机制 Figure 2 Catalytic mechanisms for β-N-acetylhexosaminidases. A: Partial result of multiple alignments ofβ-N-acetylhexosaminidases. The catalytic residues are colored. B: Protein structure and catalytic residues of GH3β-N-acetylhexosaminidases. C: Protein structure and catalytic residues of GH20 β-N-acetylhexosaminidases. D:Protein structure and catalytic residues of GH84 β-N-acetylhexosaminidases. Catalytic domains are shown inyellow. |
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1.2 GH20家族β-N-乙酰氨基己糖苷酶 GH20家族中归于β-N-乙酰氨基己糖苷酶的有64种来源于真核生物,53种来源于细菌,为数量最多的β-N-乙酰氨基己糖苷酶家族。该家族的酶对β-N-乙酰氨基葡萄糖苷和β-N-乙酰氨基半乳糖苷都有水解活性,为双功能酶。GH20家族共有14种酶的晶体结构得到解析(表 1)[10-18],自1996年首例细菌粘质沙雷氏菌源的β-N-乙酰氨基己糖苷酶晶体结构的报道以来,目前共有10种来自于细菌,而真核生物中只有人源的β-N-乙酰氨基己糖苷酶HexA(αβ)[19]和HexB(ββ)[20]、昆虫[21]以及米曲霉来源的4种酶的晶体结构解析。GH20家族β-N-乙酰氨基己糖苷酶采用底物辅助型的反应机制(图 1-C),该家族的酶都有一对高度保守的催化氨基酸对Glu-Asp,Glu为酸碱催化氨基酸,Asp为底物辅助氨基酸,这2个氨基酸位于同一个loop结构上(图 2-A和C)。大多数酶蛋白具有由芳香族氨基酸组成的疏水作用腔,可能用以把底物放置于活性位点处。2009年,Sumida等对来自类芽孢杆菌β-N-乙酰氨基己糖苷酶Hex1的C端缺失蛋白(Hex1-ΔC)与底物乙酰氨基葡萄糖(GlcNAc)和乙酰氨基半乳糖(GalNAc)等的共晶体进行了结构解析,Hex1-ΔC由2个结构域组成,分别是N端结构域和催化域,N端结构域包含2个长的α螺旋和7个β折叠,催化域为(β/α)8桶状的催化结构(图 2-C)。在催化域中,Trp352、Trp370和Trp441被认为可以将β-GlcNAc放置在活性位点处,Glu321与β-GlcNAc和β-GalNAc的O1形成氢键,推测Glu321为酸碱催化氨基酸,Asp322和Tyr395决定了2位乙酰氨基的方向,Trp410与糖环有堆叠的相互作用[10]。
对GH20家族的蛋白晶体结构分析发现,该家族中细菌源的β-N-乙酰氨基己糖苷酶结构上具有丰富的多样性,具有2-4个结构域。天蓝色链霉菌(Streptomyces coelicolor)和灰色链霉菌(Streptomyces plicatus)的β-N-乙酰氨基己糖苷酶ScGH20和SpHex均具有双结构域,即N端结构域和催化域;肺炎双球菌(Streptococcuspneumoniae)和格氏链球菌(Streptococcus gordonii)的β-N-乙酰氨基己糖苷酶GH20C和GcnA均具有3个结构域,分别是N端结构域、催化域和C端结构域。粘质沙雷氏菌(Serratia marcescens)的β-N-乙酰氨基己糖苷酶具有4个结构域,其中结构域Ш为催化结构域。GH20家族细菌源的β-N-乙酰氨基己糖苷酶的结构有一个共同的特征,即催化域均不在N端,这一特征与GH3家族的β-N-乙酰氨基葡萄糖苷酶的催化域大多在N端是不同的。真核生物来源的β-N-乙酰氨基己糖苷酶均为二聚体,且每个亚基都为由N端结构域和催化域组成双结构域蛋白。如人源β-N-乙酰氨基己糖苷酶HexA为异质二聚体,由拓扑结构相似的α亚基和β亚基组成;HexB为同质二聚体,由2个β亚基组成;玉米螟(Ostrinia furnacalis)的OfHex1为同质二聚体,也由2个亚基组成。
1.3 GH84家族β-N-乙酰氨基葡萄糖苷酶 GH84家族中归于β-N-乙酰氨基己糖苷酶的有3种来源于细菌,1种来源于真菌,均专一性水解β-N-乙酰氨基葡萄糖苷键,其中有2种酶的晶体结构得到解析,均来自产气荚膜梭菌(表 1)[22-23]。与GH20家族相似,GH84家族也采用底物辅助型的反应机制(图 1-C),催化腔内有催化氨基酸对Asp-Asp,分别作为酸碱催化氨基酸和底物辅助氨基酸(图 2-A和D)。2006年,来自产气荚膜梭菌(Clostridium perfringens)的β-N-乙酰氨基葡萄糖苷酶NagJ的晶体结构及酶与过渡态模拟抑制剂PUGNAc的共晶体得到解析,该酶具有3个结构域,分别是N端结构域、催化域和C端结构域。N端结构域由6个β折叠和3个α螺旋组成,2个α螺旋在β折片的一侧,1个α螺旋在另一侧形成三明治结构;催化域为(β/α)8桶状结构,但缺少第7个螺旋;C端结构域是由5个螺旋组成的细长的螺旋束(图 2-D)[23]。另一个来自产气荚膜梭菌的CpNagJ与NagJ结构相同。分析发现,GH84家族的催化域与GH20家族相同,均不在N端,与GH3家族的β-N-乙酰氨基葡萄糖苷酶的催化域大多在N端不同。
2 β-N-乙酰氨基己糖苷酶的生物学功能 β-N-乙酰氨基己糖苷酶广泛存在于原核生物和真核生物中,2014年Ferrara等首次发现了古菌来源的GH116家族的β-N-乙酰氨基己糖苷酶[3]。
β-N-乙酰氨基己糖苷酶在原核生物和真核生物的生理活动中发挥了重要的生物学功能。细菌细胞壁是由肽聚糖交联而成的,GH3家族霍乱弧菌(V. cholerae)和大肠杆菌(Escherichia coli)等来源的β-N-乙酰氨基葡萄糖苷酶可以水解肽聚糖中N-乙酰葡萄糖胺和N-乙酰胞壁酸之间的糖苷键,参与细菌细胞壁的循环利用。酶水解产生的1, 6-无水-N-乙酰胞壁酸酯短肽可以诱导β-内酰胺酶表达,水解β-内酰胺类抗菌药物,使得细菌产生对β-内酰胺类抗生素的抗性,基于此科学家们致力于研究细菌β-N-乙酰氨基葡萄糖苷酶的抑制物[5, 24-25];GH20家族放线杆菌(Actinobacillusactinomycetemcomitans)来源的β-N-乙酰氨基己糖苷酶Dispersin B参与水解造成人类牙周炎的放线杆菌菌膜,该菌膜由β-1, 6键型的N-乙酰葡萄糖胺线型聚合物组成[26-27],Dispersin B的水解可以导致细胞从菌膜上脱离,从而使菌膜可以扩散到口腔的其他表面;GH84家族人类病原菌酿脓链球菌(Streptococcus pyogenes)来源的β-N-乙酰氨基葡萄糖苷酶Spy1600与人类的β-N-乙酰氨基葡萄糖苷酶的N端结构域相似并且能高效地对人类O-GlcNAc修饰的糖蛋白进行去糖基化,推测Spy1600通过该水解活性改变宿主细胞的功能[28]。真菌来源的β-N-乙酰氨基己糖苷酶在真菌细胞壁的几丁质分解体系中发挥了重要的作用,该体系包含几丁质酶和β-N-乙酰氨基己糖苷酶,这两个酶共同作用调控细胞壁几丁质的降解[29-30]。古菌来源的β-N-乙酰氨基葡萄糖苷酶目前只有一例报道,该酶属于GH116家族,推测其可能参与糖蛋白或胞外聚合物的降解和循环利用[3]。植物中的β-N-乙酰氨基己糖苷酶在大米和玉米的种子发芽过程中活性升高,水解种子贮藏蛋白上的寡糖片段,推测该类酶参与了植物种子的萌发[31-32]。昆虫细胞表达了大量β-N-乙酰氨基己糖苷酶,该类酶在昆虫不同的生理活动中都发挥了重要的作用,如在几丁质外骨骼的水解过程中,几丁质首先由几丁质酶降解为寡聚糖,然后β-N-乙酰氨基己糖苷酶再将寡聚糖降解为β-N-乙酰氨基己糖[33]。
科学家们对于β-N-乙酰氨基己糖苷酶在哺乳动物中的生物学功能研究集中在人类,该类酶的异常与影响人类健康的疾病如神经退行性疾病和癌症等密切相关。哺乳动物细胞有2种不同功能的β-N-乙酰氨基己糖苷酶,溶酶体的HexA和HexB以及细胞核与细胞质的β-N-乙酰氨基己糖苷酶。溶酶体β-N-乙酰氨基己糖苷酶属于GH20家族,是由两个单体α和β组成的二聚体,这2个单体可以组成3种不同的二聚体:HexA(αβ)、HexB(ββ)和HexS(αα),这3种同工酶都可以水解β键型的N-乙酰氨基葡萄糖和N-乙酰氨基半乳糖,其中HexA可以水解呈负电性的GM2,HexB只能水解中性糖苷,其突变伴随着神经元溶酶体中GM2神经节苷脂水平的升高,进而引发严重的甚至致命的神经退行性病变,如Tay-Sachs和Sandhoff疾病[34-36]。细胞核与细胞质的β-N-乙酰氨基葡萄糖苷酶属于GH84家族,可以专一性水解β-N-乙酰氨基葡萄糖。O-GlcNAc糖基化是β-N-乙酰氨基葡萄糖以β键连接到蛋白的Ser或Thr上,已经证实O-GlcNAc糖基化/磷酸化系统能够调节信号传导、蛋白表达、降解和靶向作用。目前,科学家们认为O-GlcNAc糖基化参与了对原癌基因及抑癌基因所编码蛋白质的修饰[37-40],显示了O-GlcNAc糖基化异常与肿瘤增殖和转移可能有关。同时还发现O-GlcNAc糖基化在大脑中含量丰富,存在于神经纤丝、微管相关蛋白和网格蛋白等蛋白中,这些蛋白的O-GlcNAc糖基化的异常与一些神经退行性疾病的发生有关,推测人脑中O-GlcNAc糖基化水平的降低可能是导致阿尔茨海默症的主要原因[41-42]。因此,科学家们希望可以利用β-N-乙酰氨基葡萄糖苷酶的选择性抑制剂,使得O-GlcNAc糖基化水平升高而磷酸化水平降低,为减缓阿尔茨海默症病情的发展提供了新的思路[43-44]。
3 β-N-乙酰氨基己糖苷酶合成寡糖的应用 糖类物质参与包括血型识别、细胞间相互作用、癌症的发生和转移、免疫应答以及细菌或病毒的粘附和入侵等多种生命活动[45-46],在这些生命过程中,寡糖与糖结合蛋白间的特异识别起着核心的作用。N-乙酰氨基己糖苷在微生物、植物和动物中均有分布,并且发挥了重要的作用。它们存在于神经节苷脂、N-糖链、O-糖链、血型相关抗原、肿瘤相关糖链、人乳寡糖和几丁质上[47-48]。目前主要通过3条途径获取重要的寡糖:从生物体中提取[49]、化学法合成[50]和酶法合成[51]。从天然物质中提取糖类化合物受到原料来源的限制,而且通常目标化合物的含量很低,故而合成寡糖引起了科学家们的关注。化学法合成糖链通常需要多步保护和去保护步骤,最终目标化合物的得率不高,且非环境友好型。酶法合成糖类化合物具有反应条件温和、步骤简单、绿色环保和产量相对较高的优势,尤为重要的是,酶法合成糖类具有立体选择性(stereoselectivity)和区域选择性(regioselectivity)。在糖类合成中有两类酶被广泛应用,即糖基转移酶和糖苷酶[52]。糖基转移酶可以催化转糖基的反应,特异性地把糖基从糖基供体上转移到受体分子上合成具有特定糖苷键的糖类化合物。根据不同的供体,糖基转移酶催化的反应途径可以分为Leloir途径和非Leloir途径。非Leloir途径是以磷酸化的糖为供体进行转糖基反应,其应用受到一定限制。Leloir途径是以核苷酸化的糖为供体进行转糖基反应,如UDP-葡萄糖,目前针对该类型的酶研究较多。但糖基转移酶通常来源有限,不容易分离纯化和异源表达,反应需要的供体UDP-糖苷价格昂贵(糖苷酶底物pNP-β-GlcNAc,RMB¥473/100 mg;糖基转移酶底物UDP-GlcNAc,RMB¥2200/100 mg;Sigma)且制备困难,无法大量获得,反应体系需要多种辅助底物。另外该类酶催化的合成反应具有高度专一性,为“一酶一键”的反应模式,因此糖基转移酶对供体和受体具有很高的选择性,使得它在合成糖类化合物的实际应用中受到一定的限制。而某些糖苷酶具有转糖基活性或逆水解合成糖苷的活性,催化反应简单,底物特异性广泛、价格便宜且容易获取,可以以非保护的糖作为底物,不需要其他辅助因子,另外糖苷酶法合成化合物还可以使用修饰的供体或受体得到其他方法较难得到的糖结构,因此被广泛用于糖类化合物的合成。
3.1 真菌源的β-N-乙酰氨基己糖苷酶在寡糖合成中的应用 虽然β-N-乙酰氨基己糖苷酶在自然界广泛分布,但是用于糖苷合成的酶大多来源于真菌,其中多数来源于曲霉属(Aspergillus)[53-58],利用酶的转糖基活性催化的合成反应报道的较多,产率也高于少数利用酶的逆水解反应催化的合成反应。在转糖基活性催化的合成反应方面,1995年,Singh等以pNP-β-GalNAc为供体,以GlcNAc为受体,利用米曲霉(A. oryzae)来源的酶催化反应合成GalNAcβ1-4GlcNAc和GalNAcβ1-6GlcNAc,最高产率分别是72%和33%[53];同年,该课题组以pNP-β-GlcNAc为供体,以(GlcNAcβ1-4)3为受体,利用米曲霉(A. oryzae)来源的酶催化合成(GlcNAcβ1-4)3GlcNAc,该化合物是参与固氮细菌和豆科植物共生的结瘤因子的核心糖链[54];2003年,Uzawa等以6位硫酸根修饰的pNP-6-sulfo-β-GlcNAc为供体,以修饰的糖苷为受体,如Glcα-OAll、Galα-OAll和GlcNAc-OAll等,利用米曲霉(A. oryzae)来源的酶催化反应合成硫酸化二糖衍生物,如6-sulfo-GlcNAcβ1-4Glcα-OAll、6-sulfo-GlcNAcβ1-3/1-6Galα-OAll和6-sulfoGlcNAcβ1-4GlcNAc-OAll等,产率在17%以上,硫酸化糖是选择素、细菌和病毒以及其他糖受体蛋白识别结构的关键组成部分[55];2004年,Aboitiz等可分别以pNP-β-GalNAc和pNP-β-GlcNAc为供体,以GlcNAcβ1-4ManNAc为受体,利用米曲霉(A. oryzae)来源的酶催化反应分别合成GalNAcβ1-4GlcNAcβ1-4ManNAc和GlcNAcβ1-4GlcNAcβ1-4ManNAc,产率分别是41%和36%,显示是该酶一种双功能β-N-乙酰氨基己糖苷酶,该研究探索了橡胶蛋白对产物的结合模式[56];2004年,Rauvolfová等以pNP-β-GlcNAc为供体,以乳糖为受体,利用黄叉曲霉(Aspergillus flavofurcatis)来源的酶催化反应合成Galβ1-4Glcβ1-1GlcNAc和Galβ1-4Glcα1-1GlcNAc,产率分别是10%和9%,该研究旨在合成稀有的非还原性糖[57];2005,Lakshmanan等以pNP-β-GlcNAc为供体,以GlcNAcβNHAc为受体,利用米曲霉(A. oryzae)来源的酶催化反应合成GlcNAcβ1-4GlcNAcβNHAc,产率为17%,产物可作为N-glycan的模拟寡糖[58]。其他真菌源的还有踝节菌属(Talaromyces)[59-60]、青霉属(Penicillium)[61-62]和木霉属(Trichoderma)[63]的β-N-乙酰氨基己糖苷酶。2011年,Bojarová等以6位修饰的pNP-β-GlcNAc为供体,以GlcNAc为受体,利用黄色蠕形霉(Talaromyces flavus)来源的酶催化合成非还原端6位修饰的GlcNAcβ1-4GlcNAc,产率为23%和33%,合成的产物用以研究其在免疫反应中的作用[59];2010年,Slámová等以4位脱氧的pNP-4-deoxy-β-GlcNAc为供体,以GlcNAc为受体,利用黄色蠕形霉(T. flavus)来源的酶催化合成非还原端4-deoxy-GlcNAcβ1-4GlcNAc、4-deoxy-GlcNAcβ1-6GlcNAc和4-deoxy-GlcNAcβ1-6-4-deoxy-GlcNAc-β-pNP,产率分别为7%、6%和14%,阐明了脱氧供体对β-N-乙酰氨基己糖苷酶催化的转糖基反应的影响并探索了酶法合成脱氧二糖的方法[60]。2001年,Hu?áková等以6位乙酰化的pNP-6-acetyl-β-GlcNAc为供体,以GlcNAc为受体,利用巴西青霉(Penicillium brasilianum)来源的酶催化生成乙酰化修饰的6-acetylGlcNAcβ1-4GlcNAc,产率为21%[61];2003年,Weignerová等以pNP-β-GalNAc为供体,以GlcNAc或GalNAc为受体,利用草酸青霉(Penicilliumoxalicum)来源的酶催化合成GalNAcβ1-6GalNAc、GalNAcβ1-6GlcNAc和GalNAcβ1-4GlcNAc,产率分别为87%、19%和26.5%[62];2004年,Nieder等发现哈茨木霉(Trichoderma harzianum)来源的酶可分别以pNP-β-GalNAc和pNP-β-GlcNAc为供体,以UDP-GlcNAc为受体,催化反应分别合成GalNAcβ1-4GlcNAcα1-UDP和GlcNAc-GlcNAcUDP(键型没鉴定),产率分别是22%和1.9%,显示是一种双功能β-N-乙酰氨基己糖苷酶,该研究拓展了酶法合成的核苷糖的方法[63]。
在逆水解活性催化的合成反应方面,2003年Matsuo等将米曲霉(A. oryzae)来源的β-N-乙酰氨基己糖苷酶以GlcNAc和乳糖为底物,逆水解反应4 d合成GlcNAcβ1-3Galβ1-4Glc和GlcNAcβ1-6Galβ1-4Glc,产率分别为0.36%和0.72%,产物为重要的N-乙酰氨基己糖苷化的乳糖[64];2004年,Rauvolfova等将绳状青霉(Penicillium funiculosum)来源的酶以GlcNAc为底物,逆水解反应8 d合成GlcNAcβ1-3GlcNAc、GlcNAcβ1-4GlcNAc和GlcNAcβ1-6GlcNAc,产率分别为3.8%、1.7%和10.0%[65];2005年,Lakshmanan等将米曲霉(A. oryzae)来源的酶以GlcNAc为供体,以GlcNAcβNHCOCH3和GlcNAcβNHCOCH2CH3为受体,逆水解反应4 d合成GlcNAcβ1-6GlcNAcβNHCOCH3和GlcNAcβ1-6GlcNAcβNHCOCH2CH3,产率分别为13%和8%,产物可作为N-glycan的模拟寡糖[58]。
目前只有2例分别来自真菌米曲霉(A. oryzae)和哈茨木霉(T. harzianum)的双功能β-N-乙酰氨基己糖苷酶,可以pNP-β-GalNAc和pNP-β-GlcNAc为供体转糖基进行N-乙酰氨基己糖苷化的乳糖的合成;真菌源的β-N-乙酰氨基己糖苷酶都以离去基团较易离去的pNP-β-GalNAc或pNP-β-GlcNAc为供体进行转糖基反应;β-N-乙酰氨基己糖苷酶催化的转糖基反应合成产物的产量大都大于逆水解合成产物的产量,且逆水解反应合成糖苷的时间一般都在4 d以上。
3.2 细菌源的β-N-乙酰氨基己糖苷酶在寡糖合成中的应用 根据文献报道,目前只有4种细菌来源的β-N-乙酰氨基己糖苷酶具有转糖基活性,分别为粘质沙雷氏菌(S. marcescens)YS-1[66]、诺卡氏菌(Nocardiaorientalis)[67-68]和双歧杆菌(Bifidobacteriumbifidum)[69]。来自S. marcescens的β-N-乙酰氨基己糖苷酶以pNP-β-GlcNAc为供体,以不同醇类为受体,进行转糖基反应,转糖基效率在2.2%-26.7%,这些糖苷可能会作为功能性寡糖添加到食品中[66]。另外3种细菌来源的酶均以pNP-β-GalNAc或/和pNP-β-GlcNAc为供体,以乳糖或乳糖衍生物为受体,合成一类重要的化合物N-乙酰氨基己糖苷乳糖/乳糖衍生物。
N-乙酰氨基己糖苷乳糖具有重要的生物学功能,如GalNAcβ1-3Lac与P血型抗原糖链相似,P血型抗原糖链可以抑制呼吸道病原菌[70-71]。GlcNAcβ1-3Lac是肿瘤相关糖链的组成元件,比如KH-1腺癌抗原、N3胃肠癌抗原以及急性白血病抗原[72-73],这使得GalNAcβ1-3Lac和GlcNAcβ1-3Lac具有作为生物标记和靶向治疗的潜在应用价值。另外,GlcNAcβ1-3Lac还是人乳寡糖的核心糖链,可以作为受体合成人乳寡糖[74],作为益生元广泛应用。目前,3种细菌源的β-N-乙酰氨基己糖苷酶可以进行该类寡糖或其衍生物的合成:N. orientalis来源的2种β-N-乙酰氨基己糖苷酶以GlcNAc2为供体,分别以Galβ1-4Glc-OMe、Galβ1-4Glc-β-pNP和Galβ1-4GlcNAc-β-pNP为受体,催化转糖基反应合成GlcNAcβ1-3Galβ1-4Glc-OMe、GlcNAcβ1-3Galβ1-4Glc-β-pNP和GlcNAcβ1-3Galβ-4GlcNAc-β-pNP,产率分别是3.4%、1.9%和1.5%[67-68]。2016年本实验室应用双歧双歧杆菌(B. bifidum)BbhI以乳糖为受体,pNP-β-GalNAc和pNP-β-GlcNAc为供体,催化转糖基反应合成GalNAcβ1-3Galβ1-4Glc和GlcNAcβ1-3Galβ1-4Glc,产率分别是55.4%和44.9%[69]。
细菌源β-N-乙酰氨基己糖苷酶催化的转糖基反应的产量,遵循供体硝基苯N-乙酰氨基己糖>N-乙酰氨基己糖二聚体的规律;双歧双歧杆菌(B.bifidum) BbhI是迄今为止细菌来源唯一的双功能β-N-乙酰氨基己糖苷酶,可以pNP-β-GalNAc和pNP-β-GlcNAc为供体转糖基进行N-乙酰氨基己糖苷化乳糖的合成;目前,尚未有细菌源β-N-乙酰氨基己糖苷酶逆水解合成糖苷的报道。
3.3 宏基因组文库源β-N-乙酰氨基己糖苷酶在寡糖合成中的应用 2015年,Nyffenegger等从100000个菌落的土壤宏基因文库筛选获得GH20家族2个名为HEX1和HEX2的β-N-乙酰氨基己糖苷酶,其中HEX1基本归属于放线菌属,HEX2介于刀豆属和曲霉属之间,该两种酶都以GlcNAc2为供体,乳糖为受体,分别以2%和8%的产率合成了重要寡糖GlcNAcβ1-3Galβ1-4Glc[75]。
3.4 β-N-乙酰氨基己糖苷酶催化合成反应的区域选择性 与一般糖苷酶一样,β-N-乙酰氨基己糖苷酶催化的转糖基反应也具有严格的立体选择性(stereoselectivity),而区域选择性(regioselectivity)一般都比较灵活,这也导致了同分异构体产物的产生。糖苷酶的区域选择性与酶的来源和底物的种类有关(表 2)。米曲霉(A. oryzae)来源的β-N-乙酰氨基己糖苷酶以GlcNAc和乳糖为底物,逆水解合成GlcNAcβ1-3Galβ1-4Glc和GlcNAcβ1-6Galβ1-4Glc[64];以pNP-β-GalNAc为供体,以GlcNAc为受体,合成GalNAcβ1-4GlcNAc和GalNAcβ1-6GlcNAc[53]等。土壤宏基因组来源的β-N-乙酰氨基己糖苷酶催化以GlcNAc2为供体,乳糖为受体的转糖基反应,合成GlcNAcβ1-3Galβ1-4Glc和GlcNAcβ1-4Galβ-4Glc[75]。N. orientalis来源的酶以GlcNAc2为供体,methyl-β-lactoside为受体时,合成GlcNAcβ1-3/1-6Galβ1-4Glc-OMe和Galβ1-4(GlcNAcβ1-6) Glc-OMe;以Galβ1-4GlcNAc-β-pNP为受体时,合成GlcNAcβ1-3/1-6Galβ1-4GlcNAc-β-pNP和Galβ1-4(GlcNAcβ1-6) GlcNAc-β-pNP[67-68]。本实验室研究发现双歧双歧杆菌(B.bifidum)BbhI在以pNP-β-GalNAc和pNP-β-GlcNAc为供体、乳糖为受体时具有专一的区域选择性,对这一现象的分子对接分析结果显示乳糖中半乳糖上的3-OH与酸碱催化氨基酸形成氢键,推测该3-OH可以被酸碱催化位点激活,并被放置在合适的位置,随后对恶唑啉中间体进行攻击,从而在HexNAc和乳糖上的Gal之间形成区域选择性专一的β1-3键型。而另外3个氨基酸与乳糖中半乳糖的4和6位羟基以及葡萄糖的3和6位羟基形成氢键,可能会导致乳糖中的Gal上的3-OH而不是其他羟基朝向HexNAc的异头碳。另外乳糖处于由芳香族氨基酸形成的疏水口袋里,且与His形成π-π堆叠。这些分子特征可能都与BbhI可以专一合成β1-3键型的N-乙酰己糖胺乳糖有关[69]。
表 2. β-N-乙酰氨基己糖苷酶催化转糖基反应的供受体和产物 Table 2. Donor, acceptor, and product of β-N-acetylhexosaminidases-catalyzed transglycosylation
Enzyme source | Donor | Acceptor | Product | Reference |
Aspergillus | pNP-β-GalNAc | GlcNAc | GalNAcβ1-4GlcNAc | [53] |
GalNAcβ1-6GlcNAc | ||||
pNP-β-GlcNAc | (GlcNAcβ1-4)3 | (GlcNAcβ1-4)3GlcNAc | [54] | |
pNP-6-sulfoβGlcNAc | Glcα-OAll | 6-sulfo-GlcNAcβ1-4Glcα-OAll | [55] | |
Galα-OAll | 6-sulfo-GlcNAcβ1-3Galα-OAll | |||
6-sulfo-GlcNAcβ1-6Galα-OAll | ||||
GlcNAc-OAll | 6-sulfo-GlcNAcβ1-4GlcNAc-OAll | |||
pNP-β-GalNAc | GlcNAcβ1-4ManN | GalNAcβ1-4GlcNAcβ1-4ManNAc | [56] | |
pNP-β-GlcNAc | Ac | GlcNAcβ1-4GlcNAcβ1-4ManNAc | ||
pNP-β-GlcNAc | Galβ1-4Glc | Galβ1-4Glcβ1-1GlcNAc | [57] | |
Galβ1-4Glcα1-1GlcNAc | ||||
pNP-β-GlcNAc | GlcNAcβNHAc | GlcNAcβ1-4GlcNAcβNHAc | [58] | |
GlcNAc | Galβ1-4Glc | GlcNAcβ1-3Galβ1-4Glc | [64] | |
GlcNAcβ1-6Galβ1-4Glc | ||||
GlcNAc | GlcNAcβNHR' | GlcNAcβ1-6GlcNAcβNHR | [58] | |
Talaromyces | pNP-6R’-β-GlcNAc | GlcNAc | 6R’-GlcNAcβ1-4GlcNAc | [59] |
pNP-4deoxyβGlcNAc | GlcNAc | 4-deoxy-β-N-acetylhexosaminides | [60] | |
Penicillium | pNP-6acetylβGlcNAc | GlcNAc | 6-acetyl-GlcNAcβ1-4GlcNAc | [61] |
pNP-β-GalNAc | GalNAc | GalNAcβ1-6GalNAc | [62] | |
GlcNAc | GalNAcβ1-4GlcNAc | |||
GalNAcβ1-6GlcNAc | ||||
GlcNAc | GlcNAc | GlcNAcβ1-3GlcNAc | [65] | |
GlcNAcβ1-4GlcNAc | ||||
GlcNAcβ1-6GlcNAc | ||||
Trichoderma | pNP-β-GalNAc | UDP-GlcNAc | GalNAcβ1-4GlcNAcα1-UDP | [63] |
pNP-β-GlcNAc | GlcNAc-GlcNAc-UDP | |||
Serratia | pNP-β-GlcNAc | alcohols | Sugaralcohols | [66] |
sugar alcohols | ||||
Nocardia | GlcNAc2 | Galβ1-4Glc- | GlcNAcβ1-3Galβ1-4Glc-OMe | [68] |
OMe | GlcNAcβ1-6Galβ1-4Glc-OMe | |||
Galβ1-4(GlcNAcβ1-6) Glc-OMe | ||||
Galβ1-4Glc-β- | GlcNAcβ1-3Galβ1-4Glc-β-pNP | |||
pNP | GlcNAcβ1-6Galβ1-4Glc-β-pNP | |||
Galβ1-4(GlcNAcβ1-6) Glc-β-pNP | ||||
Galβ1-4GlcNAc-β- | GlcNAcβ1-3Galβ1-4GlcNAcβpNP | [67] | ||
pNP | GlcNAcβ1-6Galβ1-4GlcNAcβpNP | |||
Galβ1-4(GlcNAcβ1-6) GlcNAcβpNP | ||||
Bifidobacterium | pNP-β-GalNAc | Galβ1-4Glc | GalNAcβ1-3Galβ1-4Glc | [69] |
pNP-β-GlcNAc | GlcNAcβ1-3Galβ1-4Glc | |||
Soil-derived | GlcNAc2 | Galβ1-4Glc | GlcNAcβ1-3Galβ1-4Glc | [75] |
metagenomic library | GlcNAcβ1-4Galβ1-4Glc |
表选项
4 展望 β-N-乙酰氨基己糖苷酶在生命体内具有重要的生物学功能,其异常与很多人类的重大疾病相关。对该酶的研究特别是相关抑制剂的进一步探索将更好地理解其在生物体中发挥的功能以及对疾病的诊断和治疗意义重大。
当今对于寡糖的研究和应用发展迅速,多种寡糖作为医药和功能性食品基料已广泛应用。具有特定结构的重要寡糖的大量合成及其功能阐明将为疾病提供重要的检测和治疗方法。β-N-乙酰氨基己糖苷酶催化的转糖基反应正在成为酶法合成β-N-乙酰氨基己糖苷的主要方式。因此,对新酶的基因挖掘、分子改造以获得具有不同区域选择性及高效转糖基活性的酶,应用于糖类合成,将促进糖生物学的发展和糖产品的研发。
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