X-ray structure and characterization of a probiotic Lactobacillus rhamnosus Probio-M9 L-rhamnose isomerase,Applied Microbiology and Biotechnology

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X-ray structure and characterization of a probiotic Lactobacillus rhamnosus Probio-M9 L-rhamnose isomerase
Applied Microbiology and Biotechnology ( IF 3.9 ) Pub Date : 2024-03-02 , DOI: 10.1007/s00253-024-13075-9
Hiromi Yoshida _{1,

2} , Naho Yamamoto ₃ , Lin Hai Kurahara ₄ , Ken Izumori _{2,

3} , Akihide Yoshihara _{2,

3}

Affiliation

Abstract

A recombinant L-rhamnose isomerase (L-RhI) from probiotic Lactobacillus rhamnosus Probio-M9 (L. rhamnosus Probio-M9) was expressed. L. rhamnosus Probio-M9 was isolated from human colostrum and identified as a probiotic lactic acid bacterium, which can grow using L-rhamnose. L-RhI is one of the enzymes involved in L-rhamnose metabolism and catalyzes the reversible isomerization between L-rhamnose and L-rhamnulose. Some L-RhIs were reported to catalyze isomerization not only between L-rhamnose and L-rhamnulose but also between D-allulose and D-allose, which are known as rare sugars. Those L-RhIs are attractive enzymes for rare sugar production and have the potential to be further improved by enzyme engineering; however, the known crystal structures of L-RhIs recognizing rare sugars are limited. In addition, the optimum pH levels of most reported L-RhIs are basic rather than neutral, and such a basic condition causes non-enzymatic aldose-ketose isomerization, resulting in unexpected by-products. Herein, we report the crystal structures of L. rhamnosus Probio-M9 L-RhI (LrL-RhI) in complexes with L-rhamnose, D-allulose, and D-allose, which show enzyme activity toward L-rhamnose, D-allulose, and D-allose in acidic conditions, though the activity toward D-allose was low. In the complex with L-rhamnose, L-rhamnopyranose was found in the catalytic site, showing favorable recognition for catalysis. In the complex with D-allulose, D-allulofuranose and ring-opened D-allulose were observed in the catalytic site. However, bound D-allose in the pyranose form was found in the catalytic site of the complex with D-allose, which was unfavorable for recognition, like an inhibition mode. The structure of the complex may explain the low activity toward D-allose.

Key points

• Crystal structures of LrL-RhI in complexes with substrates were determined.

• LrL-RhI exhibits enzyme activity toward L-rhamnose, D-allulose, and D-allose.

• The LrL-RhI is active in acidic conditions.

中文翻译：

益生菌鼠李糖乳杆菌 Probio-M9 L-鼠李糖异构酶的 X 射线结构和表征

抽象的

表达了来自益生菌鼠李糖乳杆菌Probio-M9 ( L. rhamnosus Probio-M9) 的重组 L-鼠李糖异构酶 (L-RhI)。 L. rhamnosus Probio-M9 从人初乳中分离出来，并被鉴定为益生乳酸菌，可以利用 L-鼠李糖生长。 L-RhI是参与L-鼠李糖代谢的酶之一，催化L-鼠李糖和L-鼠李酮糖之间的可逆异构化。据报道，一些L-RhIs不仅能催化L-鼠李糖和L-鼠李酮糖之间的异构化，而且还能催化D-阿洛酮糖和D-阿洛糖（被称为稀有糖）之间的异构化。这些L-RhIs对于稀有糖生产来说是有吸引力的酶，并且有潜力通过酶工程进一步改进；然而，已知的 L-RhIs 识别稀有糖的晶体结构是有限的。此外，大多数报道的L-RhIs的最佳pH水平是碱性而不是中性，并且这种碱性条件会导致非酶促醛糖-酮糖异构化，从而产生意想不到的副产物。在此，我们报道了鼠李糖乳杆菌Probio-M9 L-RhI (LrL-RhI) 与 L-鼠李糖、D-阿洛酮糖和 D-阿洛糖复合物的晶体结构，其显示出对 L-鼠李糖、D-阿洛酮糖的酶活性和 D-阿洛糖在酸性条件下，尽管对 D-阿洛糖的活性较低。在与L-鼠李糖的复合物中，在催化位点发现了L-吡喃鼠李糖，表现出良好的催化识别性。在与D-阿洛酮糖的复合物中，在催化位点观察到D-阿洛酮糖和开环的D-阿洛酮糖。然而，在与D-阿洛糖复合物的催化位点中发现了吡喃糖形式的结合D-阿洛糖，这不利于识别，就像抑制模式一样。该复合物的结构可以解释 D-阿洛糖的低活性。