Lacto-N-biosidase Encoded by a Novel Gene of Bifidobacterium longum Subspecies longum Shows Unique Substrate Specificity and Requires a Designated Chaperone for Its Active Expression
Lacto-N-biosidase Encoded by a Novel Gene of Bifidobacterium longum Subspecies longum Shows Unique Substrate Specificity and Requires a Designated Chaperone for Its Active Expression
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DOI：
10.1074/jbc.m113.484733
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发表时间：
2013-08-30
影响因子：
4.8
通讯作者：
Katayama, Takane
中科院分区：
文献类型：
--
作者：
Sakurama, Haruko;Kiyohara, Masashi;Katayama, Takane
婴儿肠道相关双歧杆菌具有同化人乳低聚糖的特定酶组，而乳酸-N-生物苷酶(LNBase)是将人乳低聚糖的主要成分半乳糖β1-3GlcNAcβ1-3Galβ1-4Glc降解为半乳糖-N-二糖(Galβ1-3GlcNAc)和乳糖的关键酶。我们以前已经在双歧杆菌和一些长双歧杆菌亚种中发现了LNBase活性。Long um(B.long um)。随后，我们从双歧杆菌中分离到一个糖苷水解酶家族20(Gh20)LNBase；然而，长杆菌LNBase(+)菌株的基因组中不包含gh20 LNBase同源物。在此，我们揭示了BLLJ_1505和BLLJ_1506是由B.long um JCM1217的LNBase组成的。这些基因产物分别命名为LnbX和LnbY，与之前描述的蛋白质没有序列相似之处。纯化的酶仅由LnbX组成，通过保留N-四糖中的GlcNAcβ1-3Gal键、L-N-岩藻五糖I(Fucα1-2Gal-β1-3GlcNAcβ1-3Gal-β1-4Glc)和唾液酸-N-四糖a(Neu5Acα2-3Galβ1-3GlcNAcβ1-3Gal-β1-4Gal)，后两者不被gh20 LNBase所水解。在所研究的显色底物中，该酶作用于对硝基苯基(PNP)-β-乳酸-N-生物苷I(Galβ1-3-GlcNAcβ-PNP)和GalNAcβ1-3GlcNAcβ-pNP。GalNAcβ1-3-GlcNAcβ1-3-GlcNAcβ键存在于α-营养不良聚糖的O-甘露糖基中。因此，该酶可作为一种新的检测糖链结构的工具。体外复性实验表明，LnbX的正确折叠需要LnbY和金属离子(Ca~(2+)和Mg~(2+))。LnbX和LnbY同源物只在双歧杆菌、长双歧杆菌和少数肠道微生物中发现，这表明这些蛋白质是在特殊的生态位中进化的。
Infant gut-associated bifidobacteria possess species-specific enzymatic sets to assimilate human milk oligosaccharides, and lacto-N-biosidase (LNBase) is a key enzyme that degrades lacto-N- tetraose (Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc), the main component of human milk oligosaccharides, to lacto-N-biose I (Gal beta 1-3GlcNAc) and lactose. We have previously identified LNBase activity in Bifidobacterium bifidum and some strains of Bifidobacterium longum subsp. longum (B. longum). Subsequently, we isolated a glycoside hydrolase family 20 (GH20) LNBase from B. bifidum; however, the genome of the LNBase(+) strain of B. longum contains no GH20 LNBase homolog. Here, we reveal that locus tags BLLJ_1505 and BLLJ_1506 constitute LNBase from B. longum JCM1217. The gene products, designated LnbX and LnbY, respectively, showed no sequence similarity to previously characterized proteins. The purified enzyme, which consisted of LnbX only, hydrolyzed via a retaining mechanism the GlcNAc beta 1-3Gal linkage in lacto-N-tetraose, lacto-N-fucopentaose I (Fuc alpha 1-2Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc), and sialyllacto-N-tetraose a (Neu5Ac alpha 2-3Gal beta 1-3GlcNAc beta 1-3-Gal beta 1-4Gal); the latter two are not hydrolyzed by GH20 LNBase. Among the chromogenic substrates examined, the enzyme acted on p-nitrophenyl (pNP)-beta-lacto-N-bioside I (Gal beta 1-3-GlcNAc beta-pNP) and GalNAc beta 1-3GlcNAc beta-pNP.GalNAc beta 1-3-GlcNAc beta linkage has been found in O-mannosyl glycans of alpha-dystroglycan. Therefore, the enzyme may serve as a new tool for examining glycan structures. In vitro refolding experiments revealed that LnbY and metal ions (Ca2+ and Mg2+) are required for proper folding of LnbX. The LnbX and LnbY homologs have been found only in B. bifidum, B. longum, and a few gut microbes, suggesting that the proteins have evolved in specialized niches.