Structural Modelling and Structure‐Function Analysis of Zymomonas mobilis Levansucrase

Levansucrases are bacterial enzymes which produce fructan polymers from sucrose via hydrolysis and transfructosylation activities. These polymers; levan and fructooligosaccharides are valuable for food and pharmaceutical industries. Levansucrases from Gram‐positive bacteria such as Bacillus subtilis tend to produce levan, while those from Gram‐negative bacteria preferentially produce fructooligosaccharides. Zymomonas mobilis is an efficient levansucrase producer and its extracellular levansucrase can produce both fructooligosaccharides and levan depending on the reaction parameters. In this study, the structure of Z. mobilis levansucrase was modeled in order to help to understand the structure‐function relationship of the enzyme. Furthermore, amino acids previously reported to be important for levansucrase activity were mapped on the model. The structural model presents a five‐bladed propeller with a deep, negatively charged central pocket, similar to other bacterial levansucrases. Mapping showed that amino acids which previously reported to affect fructan length are located on the periphery of the structure covering the active site central pocket. Thus it is showed that, for the first time, that hydrolysis and transfructosylation reactions are catalyzed on different parts of Z. mobilis levansucrase structure. The structural location of the critical amino acids will pave the way to identify other residues which control fructan length by site directed mutagenesis without altering the overall fold of the enzyme.

Zymomonas mobilis Levansükrazının Yapısal Modellemesi ve Yapı‐Fonksiyon Analizi

Bakteriyal enzimler olan levansükrazlar, hidroliz ve transfruktosilasyon aktiviteleri ile sakkarozdan fruktan polimer oluşumunu katalizlerler. Bu polimerler; levan ve fruktooligosakkaritler, gıda ve ilaç endüstrileri için değerlidir. Bacillus subtilis gibi Gram‐pozitif bakterilerden elde edilen levansükrazlar levan üretme eğilimindeyken, Gram‐negatif bakteriler tercihen fruktooligosakkaritleri üretirler. Zymomonas mobilis etkili bir levansükraz üreticisi olup, hücre dışı levansükraz enzimi, tepkime parametrelerine bağlı olarak hem fruktooligosakkaritleri hem de levanı üretebilir. Bu çalışmada, enzimin yapı‐ fonksiyon ilişkisini anlamaya yardımcı olmak için Z. mobilis levansükrazın yapısı modellenmiş ve daha önce enzim aktivitesi için önemli olduğu bildirilen amino asitler model üzerinde haritalanmıştır. Elde edilen yapısal model, diğer bakteriyel levansükrazlara benzer şekilde derin, negatif yüklü bir merkezi cebe sahip beş bıçaklı pervane yapısına sahiptir. Amino asit haritalaması, daha önce fruktan uzunluğunu etkilediği bildirilen amino asitlerin, aktif bölge merkez cebini çevreleyecek şekilde yapının yüzeyinde bulunduğunu göstermiştir. Böylece, bu çalışma ile ilk kez, hidroliz ve transfruktosilasyon tepkimelerinin Z. mobilis levansükraz yapısının farklı kısımlarında katalizlendiği gösterilmiştir. Kritik amino asitlerin yapısal konumunun bilinmesi, fruktan uzunluğunu kontrol eden diğer amino asitlerin mutajenez yoluyla ve protein yapısına zarar vermeden belirlenmesine yardımcı olacaktır.

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