Improved Enzymatic Production of the Fucosylated Human Milk Oligosaccharide LNFP II with GH29B α-1,3/4-l-Fucosidases

Abstract

Five GH29B α-1,3/4-l-fucosidases (EC 3.2.1.111) were investigated for their ability to catalyze the formation of the human milk oligosaccharide lacto-N-fucopentaose II (LNFP II) from lacto-N-tetraose (LNT) and 3-fucosyllactose (3FL) via transglycosylation. We studied the effect of pH on transfucosylation and hydrolysis and explored the impact of specific mutations using molecular dynamics simulations. LNFP II yields of 91 and 65% were obtained for the wild-type SpGH29^C and CpAfc2 enzymes, respectively, being the highest LNFP II transglycosylation yields reported to date. BbAfcB and BiAfcB are highly hydrolytic enzymes. The results indicate that the effects of pH and buffer systems are enzyme-dependent yet relevant to consider when designing transglycosylation reactions. Replacing Thr284 in BiAfcB with Val resulted in increased transglycosylation yields, while the opposite replacement of Val258 in SpGH29^C and Val289 CpAfc2 with Thr decreased the transfucosylation, confirming a role of Thr and Val in controlling the flexibility of the acid/base loop in the enzymes, which in turn affects transglycosylation. The substitution of an Ala residue with His almost abolished secondary hydrolysis in CpAfc2 and BbAfcB. The results are directly applicable in the enhancement of transglycosylation and may have significant implications for manufacturing of LNFP II as a new infant formula ingredient.

Keywords: acid/base residue flexibility; molecular dynamics; protein engineering; transglycosylation.

Figure 1

Transglycosylation of LNT using 3FL as donor and GH29B α-l-fucosidases. The reaction scheme is shown according to the classical Koshland double-displacement mechanism. A fucosyl-enzyme intermediate is formed in the first step of the reaction, which is shared by the two reactions that can result from it, namely, hydrolysis and transglycosylation. The formation of lacto-N-fucopentaose II (LNFP II) from 3-fucosyllactose (3FL) and lacto-N-tetraose (LNT) occurs if the LNT attacks the fucosyl-enzyme intermediate, whereas the 3FL is hydrolyzed to lactose and fucose when the acceptor is a water molecule. The LNFP II transfucosylation product can also be a substrate for the secondary hydrolysis or function as a donor substrate.

Figure 2

Transfucosylation and hydrolysis catalyzed by current GH29B α-l-fucosidases. Molar transfucosylation yields ([LNFP II]/[3FL]₀; left) and hydrolyzed fucose concentration (right) obtained in transglycosylation catalyzed by CloFuc (a), CpAfc2 (b), BbAfcB (c), SpGH29^C (d), and BiAfcB (e) using 3FL as fucosyl donor substrate and LNT as acceptor substrate (n = 2) at pH 4.5 (filled squares), pH 5.7–6.0 (filled circles), pH 6.9–7.2 (filled regular triangles), and pH 8.1–8.5 (filled inverted triangles) in universal buffer UB (solid lines), and at pH 4.4–4.5 (open squares), pH 5.9–6.1 (open circles), pH 7.3–7.4 (open regular triangles), and pH 7.8–7.9 (open inverted triangles) in SB (dashed lines). The actual pH values in reactions corresponding to setup pH are listed in Table S3. CloFuc and BbAfcB produced mixtures of LNFP II and LNFP V (68/32% at the maximum yield of WT CloFuc after 60 min of reaction in UB pH 7.2, and 91/9% at the maximum yield of WT BbAfcB after 5 min of reaction in UB pH 4.5), while all other enzymes produced LNFP II only (Figure S2).

Figure 3

Zoom of the active site in the GH29B enzymes from Bifidobacterium longum subsp. infantis, BiAfcB (a, PDB 3UES), and from Streptococcus pneumoniae TIGR4, SpGH29^C (b, PDB 6ORG). (a) BiAfcB with important residues highlighted: the nucleophile Asp and its accompanying Tyr (cyan), the general acid/base Glu (orange), the glycan-stabilizing Trp “hands” (purple), the assisting Asp (pink), the His triad (the adjacent duo in lime green, the His at the end of strand β1 is lime), and the “shovel” Trp (black). The lowest energy docking of the Lewis A antigen glycan is shown, with the GlcNAc (subsite +1*) in blue, Gal (subsite +2′) in yellow, and Fuc (subsite −1) in red. (b) The nucleophile Asp (cyan) and the general acid/base Glu (orange), and the residues targeted by protein engineering in the current study, namely, residues Val258 (blue), Ser259 (teal), Trp264 (purple), Asp257 (light pink), Ala173 (salmon), and Trp127 (dark pink).

Figure 4

Zoom of residue interactions (Thr/Val, Ser/Arg/Lys) in the active sites of GH29B fucosidases from the AlphaFold2-models, CloFuc (a), CpAfc2 (b), and BbAfcB (c), and crystal structures, SpGH29^C (d, PDB 6ORG) and BiAfcB (e, PDB 3UES). Residues Thr in BiAfcB and Val in CpAfc2 and SpGH29^C are colored in marine, Ser interacts with Asn in CpAfc2, SpGH29^C and BiAfcB are colored in teal and yellow, respectively, and Arg in CloFuc and Lys in BbAfcB are colored in limon. Hydrogen bonding within residues (or with the backbone of loop 7) is shown as dashed lines.

Figure 5

Transfucosylation and hydrolysis catalyzed by current GH29B α-l-fucosidase variants. Molar transfucosylation yields ([LNFP II]/[3FL]₀) and hydrolyzed fucose concentration obtained in transglycosylation catalyzed by the wild type and mutants: CloFuc (a), CpAfc2 (b), BbAfcB (c), SpGH29^C (d), and BiAfcB (e) using 3FL as fucosyl donor substrate and LNT as acceptor substrate (n = 2) at 40 °C and corresponding optimal pH (CloFuc and CpAfc2 at UB pH 7.2, BbAfcB at UB pH 4.5, SpGH29^C at SB pH 5.9, and BiAfcB at SB pH 7.9). The symbol coloring corresponds to the type of variant: wild type (red); Val/Thr variants affecting acid/base flexibility (olive); variants targeting Ser/Lys/Arg possibly affecting the acid/base (cyan); variants of substrate- and acid/base-interacting Trp (wine); variants targeting assisting Asp (magenta); and variants transferring the best BiAfcB variants Ala to His (navy) and Trp to Glu (orange). CloFuc and BbAfcB produced mixtures of LNFP II and LNFP V (68/32% at the maximum yield of WT CloFuc after 60 min of reaction in UB pH 7.2, and 91/9% at the maximum yield of WT BbAfcB after 5 min of reaction in UB pH 4.5), while all other enzymes produced LNFP II only (Figure S2).