Crystal structure of levansucrase from the Gram-negative bacterium Gluconacetobacter diazotrophicus

Abstract

The endophytic Gram-negative bacterium Gluconacetobacter diazotrophicus SRT4 secretes a constitutively expressed levansucrase (LsdA, EC 2.4.1.10), which converts sucrose into fructooligosaccharides and levan. The enzyme is included in GH (glycoside hydrolase) family 68 of the sequence-based classification of glycosidases. The three-dimensional structure of LsdA has been determined by X-ray crystallography at a resolution of 2.5 A (1 A=0.1 nm). The structure was solved by molecular replacement using the homologous Bacillus subtilis (Bs) levansucrase (Protein Data Bank accession code 1OYG) as a search model. LsdA displays a five-bladed beta-propeller architecture, where the catalytic residues that are responsible for sucrose hydrolysis are perfectly superimposable with the equivalent residues of the Bs homologue. The comparison of both structures, the mutagenesis data and the analysis of GH68 family multiple sequences alignment show a strong conservation of the sucrose hydrolytic machinery among levansucrases and also a structural equivalence of the Bs levansucrase Ca2+-binding site to the LsdA Cys339-Cys395 disulphide bridge, suggesting similar fold-stabilizing roles. Despite the strong conservation of the sucrose-recognition site observed in LsdA, Bs levansucrase and GH32 family Thermotoga maritima invertase, structural differences appear around residues involved in the transfructosylation reaction.

Scheme 1. Various reactions catalysed by levansucrase

Figure 1. Three-dimensional structure of LsdA

Superior (a) and lateral (b) stereo views of the five-bladed β-propeller fold. The colour is ‘ramped’ from N- (blue) to C- (red) terminus. Catalytic residues Asp¹³⁵, Asp³⁰⁹ and Glu⁴⁰¹ are shown in ball-and-stick representation. (c) Stereo view of the electron density map (contoured at 1σ level) ‘carved’ around catalytic residues and other residues involved in the hydrogen-bond (broken lines) network at the active site. These Figures were prepared with PYMOL [47].

Figure 2. LsdA (Gd_lvs) and Bs levansucrase (Bs_lvs) sequence alignment based on structural superimposition generated by TOP [20]

Secondary-structural elements were assigned using DSSP [48] and are indicated by arrows (β-strands) and squiggles (helices). These elements were labelled following the Bs levansucrase numbering in [9]. A roman numeral (I–V) is assigned to each β-sheet module, and, in each module, the β-strands are numbered with a capital letter (A–D). α- and 3₁₀-helices (labelled α and η respectively) are numbered consecutively. Strictly conserved residues are boxed. The Figure was produced with ESPript [49].

Figure 3. Multiple sequence alignment of selected fragments of GH68 family members

The sequences are divided into two groups, Gram-negative and Gram-positive levansucrases headed by LsdA (Gd_lvs) and Bs levansucrase (Bs_lvs) respectively. Protein identifiers and access codes for each sequence in the SwissProt/TrEMBL and GenBank® databases are as follows: Gd LsdA (Gd_lvs, Q43998); B. pseudomallei K96243 levansucrase (SACB_Burps, YP_110564); B. cepacia R1808 hypothetical protein (hyp_Burce, ZP_00218900); Z. mobilis levansucrase (SACB_ZYMMO, Q60114, Q06487, Q60116), and invertase (invB_ZYMMO, AAA61488); Gluconacetobacter xylinus levansucrase (LsxA_Gxyl, BAA93720); Ps. syringae pv. tomato levansucrase (SACB_Psyr1, AAO54974; SACB_Psyr2, AAO55819; SACB_Psyr3, AAO59056); Ps. syringae pv. glycinea levansucrase (SACB_PSESG, O52408; SACB_PsyrG2, AAK49952); Ps. syringae pv. phaseolicola levansucrase (SACB_PSESH, O68609); R. aquatilis levansucrase (SACB_RAHAQ, O54435); Erwinia amylovora levansucrase (SACB_ERWAM, Q46654); Ps. aurantiaca levansucrase (SACB_Paura, AAL09386); Bs levansucrase (Bs_lvs, P05655, P70984); Bacillus stearothermophilus levansucrase (SACB_BACST, P94468); Bacillus amyloliquefaciens levansucrase (SACB_BACAM, P21130); Bacillus licheniformis unnamed protein product (unk_Blich, CAF05486); Clostridium acetobutylicum levansucrase (SACB_Cace, CAC1772; SACB_Cace1, CAC1774); Bacillus sp. V230 β-fructofuranosidase (bff_BspV230, BAA32083); Pa. polymyxa levansucrase (SACB_Ppol, CAB39327); Streptococcus salivarius levansucrase (SACB_STRSL, Q55242); L. reuteri inulosucrase (inu_Lreu, AAN05575), levansucrase (SACB_Lreu, AAO14618) and unnamed protein product (unk_Lreu, CAD19335); Lactobacillus johnsonii levansucrase (SACB_Ljohn, NP_964768); Streptococcus mutans levansucrase (SACB_STRMU, P11701); Lactobacillus sanfranciscensis levansucrase (SACB_Lsanf, CAD48195); Arthrobacter sp. K-1 β-fructofuranosidase (bff_AspK1, BAB72022); Ac. naeslundii FTF (ftf_Anae, AAG09737). Secondary-structural elements were assigned using DSSP [48] and are indicated by ‘e’ (β-strands) and ‘h’ (helices). Catalytic residues Asp¹³⁵, Asp³⁰⁹ and Glu⁴⁰¹ are boxed.

Figure 4. Sucrose-binding site of LsdA

The Figure shows an overlap of the co-ordinates of the Bs levansucrase E342A-sucrose complex (Protein Data Bank accession code 1PT2) with the Gv levansucrase, LsdA. Potential hydrogen-bonding interactions are represented by broken lines. LsdA residues are bond-coloured green and Bs levansucrase are turquoise. Labels of depicted residues follow the same colour pattern.