Determination of oligosaccharide product distributions of PL7 alginate lyases by their structural elements

Abstract

Alginate lyases can be used to produce well-defined alginate oligosaccharides (AOSs) because of their specificities for AOS products. A large number of alginate lyases have been recorded in the CAZy database; however, the majority are annotated-only alginate lyases that include little information on their products, thus limiting their applications. Here, we establish a simple and experiment-saving approach to predict product distributions for PL7 alginate lyases through extensive structural biology, bioinformatics and biochemical studies. Structural study on several PL7 alginate lyases reveals that two loops around the substrate binding cleft determine product distribution. Furthermore, a database containing the loop information of all annotated-only single-domain PL7 alginate lyases is constructed, enabling systematic exploration of the association between loop and product distribution. Based on these results, a simplified loop/product distribution relationship is proposed, giving us information on product distribution directly from the amino acid sequence.

Fig. 1. Product distributions of PyAly and AlyV.

The products generated by PyAly and AlyV were analyzed by high-performance liquid chromatography (HPLC). The main products of AlyV and PyAly were trisaccharides and tetrasaccharides, respectively, showing different Dp specificities.

Fig. 2. Structural analysis of the AlyV^R91A-M8, PyAly^H125A-M8 and PyAly^H125A_Y223A-M5 complexes.

Overall structures of the a AlyV^R91A-M8 complex, c PyAly^H125A-M8 complex and d PyAly^H125A_Y223A-M5 complex. AlyV^R91A, PyAly^H125A and PyAly^H125A_Y223A are shown as cartoons in pale cyan, dark green and light blue, respectively. M8 and M5 are shown as sticks in salmon a, gray c, and yellow d, respectively. The electron density maps for M8 and M5 (contour level 1σ) are shown above the overall structures of the complexes. b Hydrogen bonding interactions between AlyV^R91A and the sugar units at subsites -1 and -2. The residues involved in substrate binding are shown in sticks, and hydrogen bonds are indicated with blue dashed lines. e Structural comparison of the PyAly^H125A-M8 complex with the PyAly^H125A_Y223A-M5 complex. f Hydrogen bonding interactions between PyAly and the sugar units at subsites -3 to +3. The residues involved in substrate binding are shown in stick, and hydrogen bonds are indicated with blue dashed lines.

Fig. 3. Sequence alignment of AlyV, PyAly, A1-II’ and CD2^AlyB.

The three conserved regions in the PL7 family are boxed in red, and two conserved catalytic residues are marked with stars. For PyAly, A1-II’ and CD2^AlyB, the residues involved in substrate binding at subsites +1 to +3 are shaded in red, and the conserved residues in the PL7 family are boxed in black. For PyAly and AlyV, the residues involved in substrate binding at subsites -1 to -3 are shaded in blue.

Fig. 4. Comparative analysis of the substrate binding profiles of PyAly, A1-II’, AlyV and AlyB revealed the relationship between the “-” section and product distribution.

a Structural comparison of the PyAly^H125A-M8 + M5 complex and the A1-II’-GGMG complex. The sugars bound in PyAly and A1-II’ are shown as sticks in yellow and gray, respectively. The substrate binding clefts of PL7 alginate lyases were divided into “+” sections and “-” sections using the cleavage site as the boundary. Similar substrate binding profiles at the “+” sections were observed. b Structural comparison of the PyAly^H125A-M8 + M5 and AlyV^R91A-M8 complexes. The sugar bound in AlyV is shown in salmon. A clear angle was observed between the two sugar units at subsites -2, indicating different substrate binding profiles in the “-” sections. c Structural analysis of the AlyB^H360A_Y466A-G9 complex. AlyB is shown in cartoon, and G9 is shown in green. The electron density map for G9 (contour level 1σ) is presented. The residues that formed hydrogen bonding interactions with subsites -3 to -1 are shown as sticks: the residues from CBM32 are in slate, and the residues from CD2^AlyB are in gray. Hydrogen bonds are indicated with blue dashed lines.

Fig. 5. Two loops in the “-” section were demonstrated to determine product distribution.

a The positions of two loops (loop1 and loop2) from AlyV and PyAly. loop1^AlyV: Pro117-Ala134; loop2^AlyV: Val183-Leu210; loop1^PyAly: Thr108-Lys117; loop2^PyAly: Arg159-Phe170. b Diagram of the constructed chimeric PyAly M1, containing loop1^AlyV and loop2^AlyV. c Product distributions of M1, M1-1, and M1-2.

Fig. 6. The relationship between two loops, the substrate binding profile and product distribution.

The monomer unit of alginate is shown in a blue circle. The endolytic PL7 alginate lyase a degraded long-chain alginate b into a mixture of oligosaccharides with different Dps c. The oligosaccharides are colored with a gradient from white (low sugar content) to blue (high sugar content), indicating different product distributions. Our study demonstrated that the “+” sections of the substrate binding clefts of PL7 alginate lyases contained several conserved residues, leading to similar substrate binding profiles. In contrast, the “-” sections of the substrate binding clefts exhibited diversity, resulting in different substrate binding profiles. Thus, the overall substrate binding profiles of PL7 alginate lyases were mainly determined by the “-” sections. Structural and biochemical analyses indicated that the diverse substrate binding profiles were attributed to the different loops (loop1 and loop2) around the “-” section. Accordingly, these two loops determined the substrate binding profiles of PL7 alginate lyases. Because the substrate binding profile directly influenced the product distribution, we associated the two loops with the product distributions. Thus, two loops in the “-” section determined product distributions.

Fig. 7. The products generated by six PL7 alginate lyases were analyzed by HPLC.

The  main products of these alginate lyases were determined by the quantity of each oligomer.

Fig. 8. The association between loop1 length and product distribution was determined.

a The approach to determine the positions of loop1 and loop2 for single-domain PL7 alginate lyases. Based on the similarities in product distributions, the 24 annotated-only PL7 alginate lyases were classified into three groups and are presented in b–d. e The association between loop1 length and product distribution. The disaccharides, trisaccharides, and oligosaccharides (DP ≥ 4) are shown in green, red and blue, respectively. The circles, squares, rhombi, and triangles represent different alginate lyases sharing the same loop1 length.