Characterization of the RokA and HexA Broad-Substrate-Specificity Hexokinases from
            <i>Bacteroides fragilis</i>
            and Their Role in Hexose and
            <i>N</i>
            -Acetylglucosamine Utilization

Brigham, Christopher J.; Malamy, Michael H.

doi:10.1128/jb.187.3.890-901.2005

Cited by 37 publications

(40 citation statements)

References 49 publications

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“…Indeed, B. fragilis extracts containing NanE catalyzed the formation of NAG from manNAc ( Fig. 4A) and only formed NAG6-P if the extracts also contained the RokA kinase, which can phosphorylate NAG (6). In ⌬rokA extracts, only NAG could be detected; there was no production of NAG6-P (data not shown).…”

Section: Resultsmentioning

confidence: 96%

“…The reaction mixture contained ATP (2 mM), NADH (0.6 mM), phosphoenolpyruvate (5 mM), 1 l of pyruvate kinase (from rabbit muscle [EC 2.7.1.40], 1,351 U of activity/ml), 5 l of lactate dehydrogenase (12,500 U/ml), and 1 l of purified RokA protein (0.75 g [6]) in an assay buffer consisting of 0.1 M Tris-HCl (pH 7.0) and 0.01 M MgCl 2 . Purified NanE His6 protein and manNAc (final concentrations ranged from 33.5 M to 13.4 mM from a 0.67 M stock solution) were then added, and the reaction was monitored by measuring the decrease in the A 340 as NAD ϩ was formed from NADH at 25°C.…”

Section: Sugar Accumulation Studies (I) [mentioning

confidence: 99%

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Sialic Acid ( N -Acetyl Neuraminic Acid) Utilization by Bacteroides fragilis Requires a Novel N -Acetyl Mannosamine Epimerase

et al. 2009

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We characterized the nanLET operon in Bacteroides fragilis, whose products are required for the utilization of the sialic acid N-acetyl neuraminic acid (NANA) as a carbon and energy source. The first gene of the operon is nanL, which codes for an aldolase that cleaves NANA into N-acetyl mannosamine (manNAc) and pyruvate. The next gene, nanE, codes for a manNAc/N-acetylglucosamine (NAG) epimerase, which, intriguingly, possesses more similarity to eukaryotic renin binding proteins than to other bacterial NanE epimerase proteins. Unphosphorylated manNAc is the substrate of NanE, while ATP is a cofactor in the epimerase reaction. The third gene of the operon is nanT, which shows similarity to the major transporter facilitator superfamily and is most likely to be a NANA transporter. Deletion of any of these genes eliminates the ability of B. fragilis to grow on NANA. Although B. fragilis does not normally grow with manNAc as the sole carbon source, we isolated a B. fragilis mutant strain that can grow on this substrate, likely due to a mutation in a NAG transporter; both manNAc transport and NAG transport are affected in this strain. Deletion of the nanE epimerase gene or the rokA hexokinase gene, whose product phosphorylates NAG, in the manNAc-enabled strain abolishes growth on manNAc. Thus, B. fragilis possesses a new pathway of NANA utilization, which we show is also found in other Bacteroides species.Many bacteria have the ability to release sialic acids from complex glycoproteins and oligosaccharides present in the media or on cell surfaces at sites of colonization or infection. To use the released sialic acids as a rich source of carbon and nitrogen for growth, bacteria must have the ability to transport these compounds into the cell and convert the nine carbon sugars into intermediates that enter the central glycolytic pathways. The utilization of N-acetyl neuraminic acid (NANA), one of the sialic acids, has been well studied in Escherichia coli (36, 37), Haemophilus spp. (1, 35), and Clostridium spp. (38), to name a few.In many microorganisms, the genes for NANA utilization are arranged in an operon that may be regulated by a repressor protein, termed NanR. A comprehensive review of the organization and composition of several prokaryotic operons involved in NANA utilization has been published (36). Many of these operons share common components, including a transport gene for NANA (nanT), a gene encoding an aldolase (nanA) that splits NANA into pyruvate and N-acetyl mannosamine (manNAc), a gene encoding a kinase activity (nanK) that phosphorylates manNAc to form manNAc 6-P and, finally, an epimerase gene (nanE) whose product converts manNAc 6-P to N-acetylglucosamine 6-P (NAG 6-P). NAG 6-P then enters the common pathway of aminosugar utilization (21). For a schematic of the NANA utilization pathway in E. coli, the current paradigm of prokaryotic NANA utilization, see Fig. 7A.Bacteroides fragilis possesses a neuraminidase activity, which can liberate free NANA from complex glycoproteins and oligosaccharides. Go...

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Section: Resultsmentioning

confidence: 96%

Section: Sugar Accumulation Studies (I) [mentioning

confidence: 99%

Sialic Acid ( N -Acetyl Neuraminic Acid) Utilization by Bacteroides fragilis Requires a Novel N -Acetyl Mannosamine Epimerase

et al. 2009

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“…While the ATCC15697 chromosome does not encode an epimerase with significant identity, ROK kinases appear in the sialic acid utilization cluster (Blon_0644) and upstream of nagA and nagB (Blon_0880) clustering with N-acylglucosamine 2-epimerase (pfam07221; Blon_0875). These kinases, and putative epimerase, may function in a similar manner to the B. fragilis system, mitigating the lack of a clearly identifiable nanK gene within the ATCC15697 chromosome (50).…”

Section: Discussionmentioning

confidence: 99%

An Infant-associated Bacterial Commensal Utilizes Breast Milk Sialyloligosaccharides

Sela

Li²,

Lerno³

et al. 2011

Journal of Biological Chemistry

177

125

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Lactating mothers secrete milk sialyloligosaccharides (MSOs) that function as anti-adhesives once provided to the neonate. Particular infant-associated commensals, such as Bifidobacterium longum subsp. infantis, consume neutral milk oligosaccharides, although their ability to utilize acidic oligosaccharides has not been assessed. Temporal glycoprofiling of acidic HMO consumed during fermentation demonstrated a single composition, with several isomers, corresponding to sialylated lacto-Ntetraose. To utilize MSO, B. longum subsp. infantis deploys a sialidase that cleaves ␣2-6 and ␣2-3 linkages. NanH2, encoded within the HMO catabolic cluster is up-regulated during HMO fermentation and is active on sialylated lacto-N-tetraose. These results demonstrate that commensal microorganisms do utilize MSO, a substrate that may be enriched in the distal gastrointestinal tract.Soluble oligosaccharides often exceed protein concentrations in human milk, although their biological role remains poorly defined. These heterogeneous carbohydrates consist of glucose, galactose, N-acetylglucosamine, and frequently fucose and/or N-acetyl-D-neuraminic acid (Neu5Ac or sialic acid) residues via several potential glycosidic linkages. The human milk oligosaccharide (HMO) 4 core is typically elongated from a lactose reducing end (Gal␤1-4Glc) with iterative Gal␤1-3/ 4GlcNAc units to compose linear or branched oligosaccharides with a degree of polymerization Ն 4. ␣1-2/3/4 Fucosylation adds structural complexity and may shield the HMO backbone from exo-glycosidase digestion. Similarly, enzymatic degradation of acidic HMOs or milk sialyloligosaccharides (MSOs) requires cleavage of terminal ␣2-6-and ␣2-3-linked sialyl moieties (1). Human milk is a rich source of sialylated glycans, previously determined to be over 40 structures (of over 200 total HMOs) representing nearly 16% of soluble oligosaccharide abundances (2, 3).In contrast to lactose, the structural organization of HMOs resist host digestive enzymes, thus are introduced intact to microbial communities established along the nursing infant gastrointestinal tract (GIT) (4). Accordingly, HMOs are potent molecular arbiters at the mammalian-microbial interface as they modulate epithelial surface glycan expression and may modulate systemic immune responses (5). Moreover, HMOs limit pathogen colonization by mimicking vulnerable host epitopes thus competing for microbial adhesins. A well-studied example is the inhibition of campylobacter-induced diarrhea in infants by ␣1-2 fucosylated HMO (6, 7). Likewise, sialylated glycans are bound by Helicobacter pylori and are known to mitigate Streptococcus pneumoniae adherence to the nasopharyngeal mucosa (8 -10). In addition, sialylated milk glycoproteins confer further protection by neutralizing infectious particles such as rotavirus (11).With a few exceptions, the prominence of sialic acids to eukaryotic biology emerged with the deuterostomes and thus microbial interactions denote an evolved commensal, pathogenic, or saprotrophic relationship with anim...

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“…Thus, they must have alternate ways of transporting sugars into the cell and attaching an active phosphate moiety. Recently, two broad-specificity hexokinases from B. fragilis were characterized, and their roles in hexose and NAG utilization were studied (31). These enzymes allow utilization of nutrients found in the gut (undigested dietary polysaccharides and host-derived glycoproteins) and at sites of infection (host cell surface antigens [including the Lewis antigen] and glycolipids) (31).…”

Section: Bacteroides As Friendly Commensalsmentioning

confidence: 99%

Bacteroides: the Good, the Bad, and the Nitty-Gritty

2007

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SUMMARY Summary: Bacteroides species are significant clinical pathogens and are found in most anaerobic infections, with an associated mortality of more than 19%. The bacteria maintain a complex and generally beneficial relationship with the host when retained in the gut, but when they escape this environment they can cause significant pathology, including bacteremia and abscess formation in multiple body sites. Genomic and proteomic analyses have vastly added to our understanding of the manner in which Bacteroides species adapt to, and thrive in, the human gut. A few examples are (i) complex systems to sense and adapt to nutrient availability, (ii) multiple pump systems to expel toxic substances, and (iii) the ability to influence the host immune system so that it controls other (competing) pathogens. B. fragilis, which accounts for only 0.5% of the human colonic flora, is the most commonly isolated anaerobic pathogen due, in part, to its potent virulence factors. Species of the genus Bacteroides have the most antibiotic resistance mechanisms and the highest resistance rates of all anaerobic pathogens. Clinically, Bacteroides species have exhibited increasing resistance to many antibiotics, including cefoxitin, clindamycin, metronidazole, carbapenems, and fluoroquinolones (e.g., gatifloxacin, levofloxacin, and moxifloxacin).

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Characterization of the RokA and HexA Broad-Substrate-Specificity Hexokinases from Bacteroides fragilis and Their Role in Hexose and N -Acetylglucosamine Utilization

Cited by 37 publications

References 49 publications

Sialic Acid ( N -Acetyl Neuraminic Acid) Utilization by Bacteroides fragilis Requires a Novel N -Acetyl Mannosamine Epimerase

Sialic Acid ( N -Acetyl Neuraminic Acid) Utilization by Bacteroides fragilis Requires a Novel N -Acetyl Mannosamine Epimerase

An Infant-associated Bacterial Commensal Utilizes Breast Milk Sialyloligosaccharides

Bacteroides: the Good, the Bad, and the Nitty-Gritty

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