Haruko Sakurama

Bifidobacterium longum subsp. infantis uses two different β-galactosidases for selectively degrading type-1 and type-2 human milk oligosaccharides

The breast-fed infant intestine is often colonized by particular bifidobacteria, and human milk oligosaccharides (HMOs) are considered to be bifidogenic. Recent studies showed that Bifidobacterium longum subsp. infantis can grow on HMOs as the sole carbon source. This ability has been ascribed to the presence of a gene cluster (HMO cluster-1) contained in its genome. However, the metabolism of HMOs by the organism remains unresolved because no enzymatic studies have been completed. In the present study, we characterized β-galactosidases of this subspecies to understand how the organism degrades type-1 (Galβ1-3GlcNAc) and type-2 (Galβ1-4GlcNAc) isomers of HMOs. The results revealed that the locus tag Blon_2016 gene, which is distantly located from the HMO cluster-1, encodes a novel β-galactosidase (Bga42A) with a significantly higher specificity for lacto-N-tetraose (LNT; Galβ1-3GlcNAcβ1-3Galβ1-4Glc) than for lacto-N-biose I (Galβ1-3GlcNAc), lactose (Lac) and type-2 HMOs. The proposed name of Bga42A is LNT β-1,3-galactosidase. The Blon_2334 gene (Bga2A) located within the HMO cluster-1 encodes a β-galactosidase specific for Lac and type-2 HMOs. Real-time quantitative reverse transcription-polymerase chain reaction analysis revealed the physiological significance of Bga42A and Bga2A in HMO metabolism. The organism therefore uses two different β-galactosidases to selectively degrade type-1 and type-2 HMOs. Despite the quite rare occurrence in nature of β-galactosidases acting on type-1 chains, the close homologs of Bga42A were present in the genomes of infant-gut associated bifidobacteria that are known to consume LNT. The predominance of type-1 chains in HMOs and the conservation of Bga42A homologs suggest the coevolution of these bifidobacteria with humans.

1,3-1,4-α-l-Fucosynthase That Specifically Introduces Lewis a/x Antigens into Type-1/2 Chains

Background: Regiospecific installation of α-l-fucosyl residue into glycoconjugates is quite difficult. Results: A glycosynthase mutant of 1,3-1,4-α-l-fucosidase specifically synthesized Lewis a/x trisaccharides using Galβ1–3/4GlcNAc as acceptors. Conclusion: Structural studies provide a rationale to explain the unusually strict substrate specificity exhibited by the enzyme. Significance: A new enzymatic route for specifically introducing Lewis a/x epitopes into type-1/2 chains becomes available. α-l-Fucosyl residues attached at the non-reducing ends of glycoconjugates constitute histo-blood group antigens Lewis (Le) and ABO and play fundamental roles in various biological processes. Therefore, establishing a method for synthesizing the antigens is important for functional glycomics studies. However, regiospecific synthesis of glycosyl linkages, especially α-l-fucosyl linkages, is quite difficult to control both by chemists and enzymologists. Here, we generated an α-l-fucosynthase that specifically introduces Lea and Lex antigens into the type-1 and type-2 chains, respectively; i.e. the enzyme specifically accepts the disaccharide structures (Galβ1–3/4GlcNAc) at the non-reducing ends and attaches a Fuc residue via an α-(1,4/3)-linkage to the GlcNAc. X-ray crystallographic studies revealed the structural basis of this strict regio- and acceptor specificity, which includes the induced fit movement of the catalytically important residues, and the difference between the active site structures of 1,3-1,4-α-l-fucosidase (EC 3.2.1.111) and α-l-fucosidase (EC 3.2.1.51) in glycoside hydrolase family 29. The glycosynthase developed in this study should serve as a potentially powerful tool to specifically introduce the Lea/x epitopes onto labile glycoconjugates including glycoproteins. Mining glycosidases with strict specificity may represent the most efficient route to the specific synthesis of glycosidic bonds.

Differences in the Substrate Specificities and Active-Site Structures of Two α-L-Fucosidases (Glycoside Hydrolase Family 29) from Bacteroides thetaiotaomicron

Recent studies suggest that α-L-fucosidases of glycoside hydrolase family 29 can be divided into two subfamilies based on substrate specificity and phylogenetic clustering. To explore the validity of this classification, we enzymatically characterized two structure-solved α-L-fucosidases representing the respective subfamilies. Differences in substrate specificities are discussed in relation to differences in active-site structures between the two enzymes.

Lacto-N-biosidase encoded by a novel gene of Bifidobacterium longum subspecies longum shows unique substrate specificity and requires a designated chaperone for its active expression.

Background: Phenotypically lacto-N-biosidase-positive Bifidobacterium longum JCM1217 does not possess a gene homologous to previously identified lacto-N-biosidase. Results: Hypothetical proteins BLLJ_1505 and BLLJ_1506 encode lacto-N-biosidase and its designated chaperone, respectively. Conclusion: The enzyme showed unique and unexpected substrate specificity. Significance: The enzyme is important for understanding how B. longum consumes human milk oligosaccharides and also may serve as a new tool in glycobiology. Infant gut-associated bifidobacteria possess species-specific enzymatic sets to assimilate human milk oligosaccharides, and lacto-N-biosidase (LNBase) is a key enzyme that degrades lacto-N-tetraose (Galβ1–3GlcNAcβ1–3Galβ1–4Glc), the main component of human milk oligosaccharides, to lacto-N-biose I (Galβ1–3GlcNAc) and lactose. We have previously identified LNBase activity in Bifidobacterium bifidum and some strains of Bifidobacterium longum subsp. longum (B. longum). Subsequently, we isolated a glycoside hydrolase family 20 (GH20) LNBase from B. bifidum; however, the genome of the LNBase+ strain of B. longum contains no GH20 LNBase homolog. Here, we reveal that locus tags BLLJ_1505 and BLLJ_1506 constitute LNBase from B. longum JCM1217. The gene products, designated LnbX and LnbY, respectively, showed no sequence similarity to previously characterized proteins. The purified enzyme, which consisted of LnbX only, hydrolyzed via a retaining mechanism the GlcNAcβ1–3Gal linkage in lacto-N-tetraose, lacto-N-fucopentaose I (Fucα1–2Galβ1–3GlcNAcβ1–3Galβ1–4Glc), and sialyllacto-N-tetraose a (Neu5Acα2–3Galβ1–3GlcNAcβ1–3Galβ1–4Gal); the latter two are not hydrolyzed by GH20 LNBase. Among the chromogenic substrates examined, the enzyme acted on p-nitrophenyl (pNP)-β-lacto-N-bioside I (Galβ1–3GlcNAcβ-pNP) and GalNAcβ1–3GlcNAcβ-pNP. GalNAcβ1–3GlcNAcβ linkage has been found in O-mannosyl glycans of α-dystroglycan. Therefore, the enzyme may serve as a new tool for examining glycan structures. In vitro refolding experiments revealed that LnbY and metal ions (Ca2+ and Mg2+) are required for proper folding of LnbX. The LnbX and LnbY homologs have been found only in B. bifidum, B. longum, and a few gut microbes, suggesting that the proteins have evolved in specialized niches.

Crystal Structures of a Glycoside Hydrolase Family 20 Lacto-N-biosidase from Bifidobacterium bifidum

Background: Infant gut-associated bifidobacteria possess lacto-N-biosidase, which releases lacto-N-biose I (LNB) from human milk oligosaccharides. Results: The crystal structures of lacto-N-biosidase complexed with LNB and LNB-thiazoline were determined. Conclusion: The intermediate analog complex allows the proposal of a conformational reaction coordinate. Significance: The structures of a key enzyme in the colonization of human commensal bacteria provided its structural basis and insight into the development of inhibitors. Human milk oligosaccharides contain a large variety of oligosaccharides, of which lacto-N-biose I (Gal-β1,3-GlcNAc; LNB) predominates as a major core structure. A unique metabolic pathway specific for LNB has recently been identified in the human commensal bifidobacteria. Several strains of infant gut-associated bifidobacteria possess lacto-N-biosidase, a membrane-anchored extracellular enzyme, that liberates LNB from the nonreducing end of human milk oligosaccharides and plays a key role in the metabolic pathway of these compounds. Lacto-N-biosidase belongs to the glycoside hydrolase family 20, and its reaction proceeds via a substrate-assisted catalytic mechanism. Several crystal structures of GH20 β-N-acetylhexosaminidases, which release monosaccharide GlcNAc from its substrate, have been determined, but to date, a structure of lacto-N-biosidase is unknown. Here, we have determined the first three-dimensional structures of lacto-N-biosidase from Bifidobacterium bifidum JCM1254 in complex with LNB and LNB-thiazoline (Gal-β1,3-GlcNAc-thiazoline) at 1.8-Å resolution. Lacto-N-biosidase consists of three domains, and the C-terminal domain has a unique β-trefoil-like fold. Compared with other β-N-acetylhexosaminidases, lacto-N-biosidase has a wide substrate-binding pocket with a −2 subsite specific for β-1,3-linked Gal, and the residues responsible for Gal recognition were identified. The bound ligands are recognized by extensive hydrogen bonds at all of their hydroxyls consistent with the enzymes strict substrate specificity for the LNB moiety. The GlcNAc sugar ring of LNB is in a distorted conformation near 4E, whereas that of LNB-thiazoline is in a 4C1 conformation. A possible conformational pathway for the lacto-N-biosidase reaction is discussed.

Effects of introducing negative charges into the molecular surface of thermolysin by site-directed mutagenesis on its activity and stability

Thermolysin is remarkably activated and stabilized by neutral salts, and surface charges are suggested important in its activity and stability. The effects of introducing negative charge into the molecular surface on its activity and stability are described. Seven serine residues were selected, and each of them was changed for aspartate by site-directed mutagenesis in a thermolysin mutant. In the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-l-leucine amide, the k(cat)/K(m) values of all mutants were almost similar to that of the wild-type enzyme (WT). However, those of six out of seven mutants were enhanced 17-19 times with 4 M NaCl, being slightly higher than WT. The remaining casein-hydrolyzing activities of the S53D and S65D mutants (Ser53 and Ser65 are replaced with Asp, respectively) after 30-min incubation with 10 mM CaCl(2) at 85 degrees C were 78 and 63%, being higher than those of WT (51%) and the other mutants (35-53%). S53D was stabilized with increase in the enthalpy change of activation for thermal inactivation while S65D was with decrease in the entropy change of activation. The stability of WT was enhanced by CaCl(2) and reached the level of S53D and S65D at 100 mM, suggesting that S53D and S65D might be stabilized by reinforcement of the Ca(2+)-binding structures.

β-Glucuronidase from Lactobacillus brevis useful for baicalin hydrolysis belongs to glycoside hydrolase family 30

Baicalin (baicalein 7-O-β-d-glucuronide) is one of the major flavonoid glucuronides found in traditional herbal medicines. Because its aglycone, baicalein, is absorbed more quickly and shows more effective properties than baicalin, the conversion of baicalin into baicalein by β-glucuronidase (GUS) has drawn the attention of researchers. Recently, we have found that Lactobacillus brevis subsp. coagulans can convert baicalin to baicalein. Therefore, we aimed to identify and characterize the converting enzyme from L. brevis subsp. coagulans. First, we purified this enzyme from the cell-free extracts of L. brevis subsp. coagulans and cloned its gene. Surprisingly, this enzyme was found to be a GUS belonging to glycoside hydrolase (GH) family 30 (designated as LcGUS30), and its amino acid sequence has little similarity with any GUS belonging to GH families 1, 2, and 79 that have been reported so far. We then established a high-level expression and simple purification system of the recombinant LcGUS30 in Escherichia coli. The detailed analysis of the substrate specificity revealed that LcGUS30 has strict specificity toward glycon but not toward aglycones. Interestingly, LcGUS30 prefers baicalin rather than estrone 3-(β-d-glucuronide), one of the human endogenous steroid hormones. These results indicated that L. brevis subsp. coagulans and LcGUS30 should serve as powerful tools for the construction of a safe bioconversion system for baicalin. In addition, we propose that this novel type of GUS forms a new group in subfamily 3 of GH family 30.

Biohydrogenation of C20 polyunsaturated fatty acids by anaerobic bacteria

The PUFAs include many bioactive lipids. The microbial metabolism of C18 PUFAs is known to produce their bioactive isomers, such as conjugated FAs and hydroxy FAs, but there is little information on that of C20 PUFAs. In this study, we aimed to obtain anaerobic bacteria with the ability to produce novel PUFAs from C20 PUFAs. Through the screening of ∼100 strains of anaerobic bacteria, Clostridium bifermentans JCM 1386 was selected as a strain with the ability to saturate PUFAs during anaerobic cultivation. This strain converted arachidonic acid (cis-5,cis-8,cis-11,cis-14-eicosatetraenoic acid) and EPA (cis-5,cis-8,cis-11,cis-14,cis-17-EPA) into cis-5,cis-8,trans-13-eicosatrienoic acid and cis-5,cis-8,trans-13,cis-17-eicosatetraenoic acid, giving yields of 57% and 67% against the added PUFAs, respectively. This is the first report of the isolation of a bacterium transforming C20 PUFAs into corresponding non-methylene-interrupted FAs. We further investigated the substrate specificity of the biohydrogenation by this strain and revealed that it can convert two cis double bonds at the ω6 and ω9 positions in various C18 and C20 PUFAs into a trans double bond at the ω7 position. This study should serve to open up the development of novel potentially bioactive PUFAs.

Eukaryotic-type aromatic amino acid decarboxylase from the root colonizer Pseudomonas putida is highly specific for 3,4-dihydroxyphenyl-L-alanine, an allelochemical in the rhizosphere.

Aromatic amino acid decarboxylases (AADCs) are found in various organisms and play distinct physiological roles. AADCs from higher eukaryotes have been well studied because they are involved in the synthesis of biologically important molecules such as neurotransmitters and alkaloids. In contrast, bacterial AADCs have received less attention because of their simplicity in physiology and in target substrate (tyrosine). In the present study, we found that Pseudomonas putida KT2440 possesses an AADC homologue (PP_2552) that is more closely related to eukaryotic enzymes than to bacterial enzymes, and determined the genetic and enzymic characteristics of the homologue. The purified enzyme converted 3,4-dihydroxyphenyl-l-alanine (DOPA) to dopamine with K(m) and k(cat) values of 0.092 mM and 1.8 s(-1), respectively. The enzyme was essentially inactive towards other aromatic amino acids such as 5-hydroxy-l-tryptophan, l-phenylalanine, l-tryptophan and l-tyrosine. The observed strict substrate specificity is distinct from that of any AADC characterized so far. The proposed name of this enzyme is DOPA decarboxylase (DDC). Expression of the gene was induced by DOPA, as revealed by quantitative RT-PCR analysis. DDC is encoded in a cluster together with a LysR-type transcriptional regulator and a major facilitator superfamily transporter. This genetic organization is conserved among all sequenced P. putida strains that inhabit the rhizosphere environment, where DOPA acts as a strong allelochemical. These findings suggest the possible involvement of this enzyme in detoxification of the allelochemical in the rhizosphere, and the potential occurrence of a horizontal gene transfer event between the pseudomonad and its host organism.