Solution structure of the type I polyketide synthase Pks13 from Mycobacterium tuberculosis.

BACKGROUND: Type I polyketide synthases (PKSs) are multifunctional enzymes responsible for the biosynthesis of a group of diverse natural compounds with biotechnological and pharmaceutical interest called polyketides. The diversity of polyketides is impressive despite the limited set of catalytic domains used by PKSs for biosynthesis, leading to considerable interest in deciphering their structure-function relationships, which is challenging due to high intrinsic flexibility. Among nineteen polyketide synthases encoded by the genome of Mycobacterium tuberculosis, Pks13 is the condensase required for the final condensation step of two long acyl chains in the biosynthetic pathway of mycolic acids, essential components of the cell envelope of Corynebacterineae species. It has been validated as a promising druggable target and knowledge of its structure is essential to speed up drug discovery to fight against tuberculosis. RESULTS: We report here a quasi-atomic model of Pks13 obtained using small-angle X-ray scattering of the entire protein and various molecular subspecies combined with known high-resolution structures of Pks13 domains or structural homologues. As a comparison, the low-resolution structures of two other mycobacterial polyketide synthases, Mas and PpsA from Mycobacterium bovis BCG, are also presented. This study highlights a monomeric and elongated state of the enzyme with the apo- and holo-forms being identical at the resolution probed. Catalytic domains are segregated into two parts, which correspond to the condensation reaction per se and to the release of the product, a pivot for the enzyme flexibility being at the interface. The two acyl carrier protein domains are found at opposite sides of the ketosynthase domain and display distinct characteristics in terms of flexibility. CONCLUSIONS: The Pks13 model reported here provides the first structural information on the molecular mechanism of this complex enzyme and opens up new perspectives to develop inhibitors that target the interactions with its enzymatic partners or between catalytic domains within Pks13 itself.

Molecular Basis for Extender Unit Specificity of Mycobacterial Polyketide Synthases.

Mycobacterium tuberculosis is the causative agent of the tuberculosis disease, which claims more human lives each year than any other bacterial pathogen. M. tuberculosis and other mycobacterial pathogens have developed a range of unique features that enhance their virulence and promote their survival in the human host. Among these features lies the particular cell envelope with high lipid content, which plays a substantial role in mycobacterial pathogenicity. Several envelope components of M. tuberculosis and other mycobacteria, e.g., mycolic acids, phthiocerol dimycocerosates, and phenolic glycolipids, belong to the "family" of polyketides, secondary metabolites synthesized by fascinating versatile enzymes-polyketide synthases. These megasynthases consist of multiple catalytic domains, among which the acyltransferase domain plays a key role in selecting and transferring the substrates required for polyketide extension. Here, we present three new crystal structures of acyltransferase domains of mycobacterial polyketide synthases and, for one of them, provide evidence for the identification of residues determining extender unit specificity. Unravelling the molecular basis for such specificity is of high importance considering the role played by extender units for the final structure of key mycobacterial components. This work provides major advances for the use of mycobacterial polyketide synthases as potential therapeutic targets and, more generally, contributes to the prediction and bioengineering of polyketide synthases with desired specificity.

Engineering a branching sucrase for flavonoid glucoside diversification.

Enzymatic glycosylation of flavonoids is an efficient mean to protect aglycons against degradation while enhancing their solubility, life time and, by extension, their bioavailability which is critical for most of their applications in health care. To generate a valuable enzymatic platform for flavonoid glucosylation, an α-1,2 branching sucrase belonging to the family 70 of glycoside-hydrolases was selected as template and subsequently engineered. Two libraries of variants targeting pair-wise mutations inferred by molecular docking simulations were generated and screened for quercetin glucosylation using sucrose as a glucosyl donor. Only a limited number of variants (22) were retained on the basis of quercetin conversion and product profile. Their acceptor promiscuity towards five other flavonoids was subsequently assessed, and the automated screening effort revealed variants showing remarkable ability for luteolin, morin and naringenin glucosylation with conversion ranging from 30% to 90%. Notably, naringenin and morin, a priori considered as recalcitrant compounds to glucosylation using this α-transglucosylases, could also be modified. The approach reveals the potential of small platforms of engineered GH70 α-transglucosylases and opens up the diversity of flavonoid glucosides to molecular structures inaccessible yet.

Structural Insights into the Carbohydrate Binding Ability of an α-(1→2) Branching Sucrase from Glycoside Hydrolase Family 70.

The α-(1→2) branching sucrase ΔN123-GBD-CD2 is a transglucosylase belonging to glycoside hydrolase family 70 (GH70) that catalyzes the transfer ofd-glucosyl units from sucroseto dextrans or gluco-oligosaccharides via the formation of α-(1→2) glucosidic linkages. The first structures of ΔN123-GBD-CD2 in complex withd-glucose, isomaltosyl, or isomaltotriosyl residues were solved. The glucose complex revealed three glucose-binding sites in the catalytic gorge and six additional binding sites at the surface of domains B, IV, and V. Soaking with isomaltotriose or gluco-oligosaccharides led to structures in which isomaltosyl or isomaltotriosyl residues were found in glucan binding pockets located in domain V. One aromatic residue is systematically identified at the bottom of these pockets in stacking interaction with one glucosyl moiety. The carbohydrate is also maintained by a network of hydrogen bonds and van der Waals interactions. The sequence of these binding pockets is conserved and repeatedly present in domain V of several GH70 glucansucrases known to bind α-glucans. These findings provide the first structural evidence of the molecular interaction occurring between isomalto-oligosaccharides and domain V of the GH70 enzymes.

Characterization of the First α-(1→3) Branching Sucrases of the GH70 Family.

Leuconostoc citreumNRRL B-742 has been known for years to produce a highly α-(1→3)-branched dextran for which the synthesis had never been elucidated. In this work a gene coding for a putative α-transglucosylase of the GH70 family was identified in the reported genome of this bacteria and functionally characterized. From sucrose alone, the corresponding recombinant protein, named BRS-B, mainly catalyzed sucrose hydrolysis and leucrose synthesis. However, in the presence of sucrose and a dextran acceptor, the enzyme efficiently transferred the glucosyl residue from sucrose to linear α-(1→6) dextrans through the specific formation of α-(1→3) linkages. To date, BRS-B is the first reported α-(1→3) branching sucrase. Using a suitable sucrose/dextran ratio, a comb-like dextran with 50% of α-(1→3) branching was synthesized, suggesting that BRS-B is likely involved in the comb-like dextran produced byL. citreumNRRL B-742. In addition, data mining based on the search for specific sequence motifs allowed the identification of two genes putatively coding for branching sucrases in the genome ofLeuconostoc fallaxKCTC3537 andLactobacillus kunkeeiEFB6. Biochemical characterization of the corresponding recombinant enzymes confirmed their branching specificity, revealing that branching sucrases are not only found inL. citreumspecies. According to phylogenetic analyses, these enzymes are proposed to constitute a new subgroup of the GH70 family.

Branching pattern of gluco-oligosaccharides and 1.5kDa dextran grafted by the α-1,2 branching sucrase GBD-CD2.

GBD-CD2, an engineered sucrose-acting enzyme of glycoside hydrolase family 70, transfers D-glucopyranosyl (D-Glcp) units from sucrose onto dextrans or gluco-oligosaccharides (GOS) through the formation of α-(1→2) linkages leading to branched products of interest for health, food and cosmetic applications. Structural characterization of the branched products obtained from sucrose and pure GOS of degree of polymerization (DP) 4 or DP 5 revealed that highly α-(1→2) branched and new molecular structures can be synthesized by GBD-CD2. The formation of α-(1→2) branching is kinetically controlled and can occur onto vicinal α-(1→6)-linked D-Glcp residues. To investigate the mode of branching of 1.5 kDa dextran, simulations of various branching scenarios and resistance to glucoamylase degradation were performed. Analysis of the simulation results suggests that the branching process is stochastic and indicates that the enzyme acceptor site can accommodate both linear and poly-branched acceptors. This opens the way to the design of novel enzyme-based processes yielding carbohydrate structures varying in size and resistance to hydrolytic enzymes.

Functional and structural characterization of α-(1->2) branching sucrase derived from DSR-E glucansucrase.

ΔN(123)-glucan-binding domain-catalytic domain 2 (ΔN(123)-GBD-CD2) is a truncated form of the bifunctional glucansucrase DSR-E from Leuconostoc mesenteroides NRRL B-1299. It was constructed by rational truncation of GBD-CD2, which harbors the second catalytic domain of DSR-E. Like GBD-CD2, this variant displays α-(1→2) branching activity when incubated with sucrose as glucosyl donor and (oligo-)dextran as acceptor, transferring glucosyl residues to the acceptor via a ping-pong bi-bi mechanism. This allows the formation of prebiotic molecules containing controlled amounts of α-(1→2) linkages. The crystal structure of the apo α-(1→2) branching sucrase ΔN(123)-GBD-CD2 was solved at 1.90 Å resolution. The protein adopts the unusual U-shape fold organized in five distinct domains, also found in GTF180-ΔN and GTF-SI glucansucrases of glycoside hydrolase family 70. Residues forming subsite -1, involved in binding the glucosyl residue of sucrose and catalysis, are strictly conserved in both GTF180-ΔN and ΔN(123)-GBD-CD2. Subsite +1 analysis revealed three residues (Ala-2249, Gly-2250, and Phe-2214) that are specific to ΔN(123)-GBD-CD2. Mutation of these residues to the corresponding residues found in GTF180-ΔN showed that Ala-2249 and Gly-2250 are not directly involved in substrate binding and regiospecificity. In contrast, mutant F2214N had lost its ability to branch dextran, although it was still active on sucrose alone. Furthermore, three loops belonging to domains A and B at the upper part of the catalytic gorge are also specific to ΔN(123)-GBD-CD2. These distinguishing features are also proposed to be involved in the correct positioning of dextran acceptor molecules allowing the formation of α-(1→2) branches.

In vitro fermentation of linear and alpha-1,2-branched dextrans by the human fecal microbiota.

The role of structure and molecular weight in fermentation selectivity in linear α-1,6 dextrans and dextrans with α-1,2 branching was investigated. Fermentation by gut bacteria was determined in anaerobic, pH-controlled fecal batch cultures after 36 h. Inulin (1%, wt/vol), which is a known prebiotic, was used as a control. Samples were obtained at 0, 10, 24, and 36 h of fermentation for bacterial enumeration by fluorescent in situ hybridization and short-chain fatty acid analyses. The gas production of the substrate fermentation was investigated in non-pH-controlled, fecal batch culture tubes after 36 h. Linear and branched 1-kDa dextrans produced significant increases in Bifidobacterium populations. The degree of α-1,2 branching did not influence the Bifidobacterium populations; however, α-1,2 branching increased the dietary fiber content, implying a decrease in digestibility. Other measured bacteria were unaffected by the test substrates except for the Bacteroides-Prevotella group, the growth levels of which were increased on inulin and 6- and 70-kDa dextrans, and the Faecalibacterium prausnitzii group, the growth levels of which were decreased on inulin and 1-kDa dextrans. A considerable increase in short-chain fatty acid concentration was measured following the fermentation of all dextrans and inulin. Gas production rates were similar among all dextrans tested but were significantly slower than that for inulin. The linear 1-kDa dextran produced lower total gas and shorter time to attain maximal gas production compared to those of the 70-kDa dextran (branched) and inulin. These findings indicate that dextrans induce a selective effect on the gut flora, short-chain fatty acids, and gas production depending on their length.

Synthesis of dextrans with controlled amounts of alpha-1,2 linkages using the transglucosidase GBD-CD2.

GBD-CD2 is an alpha-1,2 transglucosidase engineered from DSR-E, a glucansucrase naturally produced by Leuconostoc mesenteroides NRRL B-1299. This enzyme catalyses from sucrose, the alpha-1,2 transglucosylation of glucosyl moieties onto alpha-1,6 dextran chains. Steady-state kinetic studies showed that hydrolysis and transglucosylation reactions occurred at the early stage of the reaction in the presence of 70 kDa dextran as acceptor and sucrose. The transglucosylation reaction catalysed by GBD-CD2 follows a Ping Pong Bi Bi mechanism with a high kcat value of 970 s(-1). The amount of the synthesised alpha-1,2 side chains was found to be directly dependent on the initial molar ratio [Sucrose]/[Dextran]. Dextrans with controlled alpha-1,2 linkage contents ranging from 13% to 40% were synthesised. The procedure resulted in the production of dextrans with the highest content of alpha-1,2 linkages ever reported.