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In silico biosynthesis of virenose, a methylated deoxy-sugar unique to Coxiella burnetii lipopolysaccharide.

Flores-Ramirez G, Janecek S, Miernyk JA, Skultety L - Proteome Sci (2012)

Bottom Line: As a prelude to a full biosynthetic characterization, we present herein the results from bioinformatics-based, proteomics-supported predictions of the pathway for virenose synthesis.Both pathways require five enzymatic steps, beginning with either glucose-6-phosphate or mannose-6-phosphate.Our in silico results comprise a model for virenose biosynthesis that can be directly tested.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Rickettsiology, Institute of Virology, Slovak Academy of Sciences, Dubravska cesta, 9, Bratislava, 845 05, Slovakia. viruludo@savba.sk.

ABSTRACT

Background: Coxiella burnetii is Gram-negative bacterium responsible for the zoonosis Q-fever. While it has an obligate intracellular growth habit, it is able to persist for extended periods outside of a host cell and can resist environmental conditions that would be lethal to most prokaryotes. It is these extracellular bacteria that are the infectious stage encountered by eukaryotic hosts. The intracellular form has evolved to grow and replicate within acidified parasitophorous vacuoles. The outer coat of C. burnetii comprises a complex lipopolysaccharide (LPS) component that includes the unique methylated-6-deoxyhexose, virenose. Although potentially important as a biomarker for C. burnetii, the pathway for its biosynthesis remains obscure.

Results: The 6-deoxyhexoses constitute a large family integral to the LPS of many eubacteria. It is believed that precursors of the methylated-deoxyhexoses traverse common early biosynthetic steps as nucleotide-monosaccharides. As a prelude to a full biosynthetic characterization, we present herein the results from bioinformatics-based, proteomics-supported predictions of the pathway for virenose synthesis. Alternative possibilities are considered which include both GDP-mannose and TDP-glucose as precursors.

Conclusion: We propose that biosynthesis of the unique C. burnetii biomarker, virenose, involves an early pathway similar to that of other C-3'-methylated deoxysugars which then diverges depending upon the nucleotide-carrier involved. The alternatives yield either the D- or L-enantiomers of virenose. Both pathways require five enzymatic steps, beginning with either glucose-6-phosphate or mannose-6-phosphate. Our in silico results comprise a model for virenose biosynthesis that can be directly tested. Definition of this pathway should facilitate the development of therapeutic agents useful for treatment of Q fever, as well as allowing improvements in the methods for diagnosing this highly infectious disease.

No MeSH data available.


Related in: MedlinePlus

The proposed pathways of C. burnetii for virenose biosynthesis. The pathway might begin with either fructose-6-phosphate (1) glucose-6-phosphate (2) or mannose-6-phosphate (3). The hexose-6-phosphates are then converted to either glucose-1-phosphate (4) or manose-1-phosphate (5) respectively by a dual-specific α-D-phosphohexomutase. Next, thymidylyltransferase or guanylyltransferase generates dTDP-glucose (6) or GDP-mannose (7), respectively. The activated sugars are transformed to the common intermediates in the biosynthesis of deoxysugars dTDP-4-keto-6-deoxy-D-glucose (8) or GDP-4-keto-6-deoxy-D-mannose (9). The carbohydrates are then methylated at C3 by the product of the TylCIII gene yielding the corresponding intermediates (10, 11). Finally, the methylated TDP intermediate is reduced by a 4-ketoreductase to form TDP-D-virenose (12). In the GDP route the intermediate GDP-3-methyl-4-keto-6-deoxy-D-idose (11) is transformed by GDP-4-keto-6-deoxy-D-mannose epimerase/reductase to GDP-L-virenose (13) or it can be converted to GDP-D-virenose (15) by the activities of a 4-ketoreductase plus a 2-C’-epimerase.
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Figure 1: The proposed pathways of C. burnetii for virenose biosynthesis. The pathway might begin with either fructose-6-phosphate (1) glucose-6-phosphate (2) or mannose-6-phosphate (3). The hexose-6-phosphates are then converted to either glucose-1-phosphate (4) or manose-1-phosphate (5) respectively by a dual-specific α-D-phosphohexomutase. Next, thymidylyltransferase or guanylyltransferase generates dTDP-glucose (6) or GDP-mannose (7), respectively. The activated sugars are transformed to the common intermediates in the biosynthesis of deoxysugars dTDP-4-keto-6-deoxy-D-glucose (8) or GDP-4-keto-6-deoxy-D-mannose (9). The carbohydrates are then methylated at C3 by the product of the TylCIII gene yielding the corresponding intermediates (10, 11). Finally, the methylated TDP intermediate is reduced by a 4-ketoreductase to form TDP-D-virenose (12). In the GDP route the intermediate GDP-3-methyl-4-keto-6-deoxy-D-idose (11) is transformed by GDP-4-keto-6-deoxy-D-mannose epimerase/reductase to GDP-L-virenose (13) or it can be converted to GDP-D-virenose (15) by the activities of a 4-ketoreductase plus a 2-C’-epimerase.

Mentions: There is biochemical evidence that C. burnetii can convert glucose to pyruvate [26-28], however, genome sequence analysis of all six isolates has thus far failed to identify a hexokinase responsible for converting glucose to glucose-6-phosphate (Figure 1) or glucose-6-phosphate and 6-phosphogluconate dehydrogenases [29]. Thus, the first steps of both glycolysis and the pentose phosphate pathway appear missing [29]. This might well explain the low biosynthetic capacity and slow growth rate observed for C. burnetii. We speculate that C. burnetii phosphorylates glucose via a transphosphorylation reaction involving carbamoyl-phosphate and a phosphatidic acid phosphatase family protein encoded by CBU_1267, as described for the 9Mi/I isolate [29]. There are, of course, other as yet-poorly defined alternatives. Possibly glucose-6-phosphate (2) is obtained from the host cells. Both GDP-mannose, and fructose 6-phosphate (1) or mannose-6-phosphate (3) are potential sources of glucose-6-phosphate, invoking participation of a mannose-6-phosphate isomerase pyrophosphorylase-type or reaction (PMI-GMP; E.C. 5.3.1.8) [30] such as that found as a participant in synthesis of the capsular polysaccharide of Pseudomonas aeruginosa, Salmonella thyphimurium, and Xanthomonas campestris[31,32]. The PMI-GMP enzymes posses separate domains for the mannose isomerase (PMI) and GDP-D-mannose pyrophosphorylase (GMP) activities [33]. A zinc-binding motif and the catalytic amino acid residue R408 are both characteristic of PMI activity [34]. The GMP activity is defined in the N-terminal by the pyrophosphorylase signature sequence, GXGXR(L)-PK [34]. Based on sequence analysis and comparison to C. burnetii genome, the PMI-GMP activity might be catalyzed by the product of the CBU_0671 gene, which includes both of these signatures. It shares 45% (E value e-113), 46% (E value 3e-122), and 39% amino acid identity (E value 4e-88) with the PMI-GMP from Salmonella enterica LT2 (AAG41744.1), Escherichia coli (YP_002413091), and Helicobacter pylori (YP_626781) (Additional file 1-A).


In silico biosynthesis of virenose, a methylated deoxy-sugar unique to Coxiella burnetii lipopolysaccharide.

Flores-Ramirez G, Janecek S, Miernyk JA, Skultety L - Proteome Sci (2012)

The proposed pathways of C. burnetii for virenose biosynthesis. The pathway might begin with either fructose-6-phosphate (1) glucose-6-phosphate (2) or mannose-6-phosphate (3). The hexose-6-phosphates are then converted to either glucose-1-phosphate (4) or manose-1-phosphate (5) respectively by a dual-specific α-D-phosphohexomutase. Next, thymidylyltransferase or guanylyltransferase generates dTDP-glucose (6) or GDP-mannose (7), respectively. The activated sugars are transformed to the common intermediates in the biosynthesis of deoxysugars dTDP-4-keto-6-deoxy-D-glucose (8) or GDP-4-keto-6-deoxy-D-mannose (9). The carbohydrates are then methylated at C3 by the product of the TylCIII gene yielding the corresponding intermediates (10, 11). Finally, the methylated TDP intermediate is reduced by a 4-ketoreductase to form TDP-D-virenose (12). In the GDP route the intermediate GDP-3-methyl-4-keto-6-deoxy-D-idose (11) is transformed by GDP-4-keto-6-deoxy-D-mannose epimerase/reductase to GDP-L-virenose (13) or it can be converted to GDP-D-virenose (15) by the activities of a 4-ketoreductase plus a 2-C’-epimerase.
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Related In: Results  -  Collection

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Figure 1: The proposed pathways of C. burnetii for virenose biosynthesis. The pathway might begin with either fructose-6-phosphate (1) glucose-6-phosphate (2) or mannose-6-phosphate (3). The hexose-6-phosphates are then converted to either glucose-1-phosphate (4) or manose-1-phosphate (5) respectively by a dual-specific α-D-phosphohexomutase. Next, thymidylyltransferase or guanylyltransferase generates dTDP-glucose (6) or GDP-mannose (7), respectively. The activated sugars are transformed to the common intermediates in the biosynthesis of deoxysugars dTDP-4-keto-6-deoxy-D-glucose (8) or GDP-4-keto-6-deoxy-D-mannose (9). The carbohydrates are then methylated at C3 by the product of the TylCIII gene yielding the corresponding intermediates (10, 11). Finally, the methylated TDP intermediate is reduced by a 4-ketoreductase to form TDP-D-virenose (12). In the GDP route the intermediate GDP-3-methyl-4-keto-6-deoxy-D-idose (11) is transformed by GDP-4-keto-6-deoxy-D-mannose epimerase/reductase to GDP-L-virenose (13) or it can be converted to GDP-D-virenose (15) by the activities of a 4-ketoreductase plus a 2-C’-epimerase.
Mentions: There is biochemical evidence that C. burnetii can convert glucose to pyruvate [26-28], however, genome sequence analysis of all six isolates has thus far failed to identify a hexokinase responsible for converting glucose to glucose-6-phosphate (Figure 1) or glucose-6-phosphate and 6-phosphogluconate dehydrogenases [29]. Thus, the first steps of both glycolysis and the pentose phosphate pathway appear missing [29]. This might well explain the low biosynthetic capacity and slow growth rate observed for C. burnetii. We speculate that C. burnetii phosphorylates glucose via a transphosphorylation reaction involving carbamoyl-phosphate and a phosphatidic acid phosphatase family protein encoded by CBU_1267, as described for the 9Mi/I isolate [29]. There are, of course, other as yet-poorly defined alternatives. Possibly glucose-6-phosphate (2) is obtained from the host cells. Both GDP-mannose, and fructose 6-phosphate (1) or mannose-6-phosphate (3) are potential sources of glucose-6-phosphate, invoking participation of a mannose-6-phosphate isomerase pyrophosphorylase-type or reaction (PMI-GMP; E.C. 5.3.1.8) [30] such as that found as a participant in synthesis of the capsular polysaccharide of Pseudomonas aeruginosa, Salmonella thyphimurium, and Xanthomonas campestris[31,32]. The PMI-GMP enzymes posses separate domains for the mannose isomerase (PMI) and GDP-D-mannose pyrophosphorylase (GMP) activities [33]. A zinc-binding motif and the catalytic amino acid residue R408 are both characteristic of PMI activity [34]. The GMP activity is defined in the N-terminal by the pyrophosphorylase signature sequence, GXGXR(L)-PK [34]. Based on sequence analysis and comparison to C. burnetii genome, the PMI-GMP activity might be catalyzed by the product of the CBU_0671 gene, which includes both of these signatures. It shares 45% (E value e-113), 46% (E value 3e-122), and 39% amino acid identity (E value 4e-88) with the PMI-GMP from Salmonella enterica LT2 (AAG41744.1), Escherichia coli (YP_002413091), and Helicobacter pylori (YP_626781) (Additional file 1-A).

Bottom Line: As a prelude to a full biosynthetic characterization, we present herein the results from bioinformatics-based, proteomics-supported predictions of the pathway for virenose synthesis.Both pathways require five enzymatic steps, beginning with either glucose-6-phosphate or mannose-6-phosphate.Our in silico results comprise a model for virenose biosynthesis that can be directly tested.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Rickettsiology, Institute of Virology, Slovak Academy of Sciences, Dubravska cesta, 9, Bratislava, 845 05, Slovakia. viruludo@savba.sk.

ABSTRACT

Background: Coxiella burnetii is Gram-negative bacterium responsible for the zoonosis Q-fever. While it has an obligate intracellular growth habit, it is able to persist for extended periods outside of a host cell and can resist environmental conditions that would be lethal to most prokaryotes. It is these extracellular bacteria that are the infectious stage encountered by eukaryotic hosts. The intracellular form has evolved to grow and replicate within acidified parasitophorous vacuoles. The outer coat of C. burnetii comprises a complex lipopolysaccharide (LPS) component that includes the unique methylated-6-deoxyhexose, virenose. Although potentially important as a biomarker for C. burnetii, the pathway for its biosynthesis remains obscure.

Results: The 6-deoxyhexoses constitute a large family integral to the LPS of many eubacteria. It is believed that precursors of the methylated-deoxyhexoses traverse common early biosynthetic steps as nucleotide-monosaccharides. As a prelude to a full biosynthetic characterization, we present herein the results from bioinformatics-based, proteomics-supported predictions of the pathway for virenose synthesis. Alternative possibilities are considered which include both GDP-mannose and TDP-glucose as precursors.

Conclusion: We propose that biosynthesis of the unique C. burnetii biomarker, virenose, involves an early pathway similar to that of other C-3'-methylated deoxysugars which then diverges depending upon the nucleotide-carrier involved. The alternatives yield either the D- or L-enantiomers of virenose. Both pathways require five enzymatic steps, beginning with either glucose-6-phosphate or mannose-6-phosphate. Our in silico results comprise a model for virenose biosynthesis that can be directly tested. Definition of this pathway should facilitate the development of therapeutic agents useful for treatment of Q fever, as well as allowing improvements in the methods for diagnosing this highly infectious disease.

No MeSH data available.


Related in: MedlinePlus