D-Xylose Degradation Pathway in the Halophilic Archaeon Haloferax volcanii

Ulrike Johnsen,Michael Dambeck,Henning Zaiss,Tobias Fuhrer,Jörg Soppa,Uwe Sauer,Peter Schönheit

doi:10.1074/jbc.m109.003814

Abstract

The pathway of D-xylose degradation in archaea is unknown. In a previous study we identified in Haloarcula marismortui the first enzyme of xylose degradation, an inducible xylose dehydrogenase (Johnsen, U., and Schönheit, P. (2004) J. Bacteriol. 186, 6198-6207). Here we report a comprehensive study of the complete D-xylose degradation pathway in the halophilic archaeon Haloferax volcanii. The analyses include the following: (i) identification of the degradation pathway in vivo following (13)C-labeling patterns of proteinogenic amino acids after growth on [(13)C]xylose; (ii) identification of xylose-induced genes by DNA microarray experiments; (iii) characterization of enzymes; and (iv) construction of in-frame deletion mutants and their functional analyses in growth experiments. Together, the data indicate that D-xylose is oxidized exclusively to the tricarboxylic acid cycle intermediate alpha-ketoglutarate, involving D-xylose dehydrogenase (HVO_B0028), a novel xylonate dehydratase (HVO_B0038A), 2-keto-3-deoxyxylonate dehydratase (HVO_B0027), and alpha-ketoglutarate semialdehyde dehydrogenase (HVO_B0039). The functional involvement of these enzymes in xylose degradation was proven by growth studies of the corresponding in-frame deletion mutants, which all lost the ability to grow on d-xylose, but growth on glucose was not significantly affected. This is the first report of an archaeal D-xylose degradation pathway that differs from the classical D-xylose pathway in most bacteria involving the formation of xylulose 5-phosphate as an intermediate. However, the pathway shows similarities to proposed oxidative pentose degradation pathways to alpha-ketoglutarate in few bacteria, e.g. Azospirillum brasilense and Caulobacter crescentus, and in the archaeon Sulfolobus solfataricus.

Highlights

In these organisms L-arabinose is oxidatively degraded to ␣-ketoglutarate, an intermediate of the tricarboxylic acid cycle, via the activities of L-arabinose dehydrogenase, L-arabinolactonase, and two successive dehydration reactions forming 2-keto-3-deoxy-L-arabinoate and ␣-ketoglutarate semialdehyde; the latter compound is further oxidized to ␣-ketoglutarate via NADPϩ-specific ␣-ketoglutarate semialdehyde dehydrogenase (KGSADH)
The degradation pathway of D-xylose in H. volcanii was analyzed as follows: (i) in vivo 13C-labeling experiments following growth on [13C]xylose; (ii) enzyme measurements in cell extracts; (iii) DNA microarray analyses to identify xylose-inducible genes; (iv) purification and characterization of enzymes involved in xylose degradation; and (v) proof of functional involvement of genes and enzymes in xylose degradation by analyzing the corresponding in-frame deletion mutants
D-Xylose might be directly converted to ␣-ketoglutarate in analogy to arabinose oxidation pathways in Azospirillum brasilense and Sulfolobus solfataricus, or it might be the converted to pyruvate and glycolaldehyde as proposed for few Pseudomonas and Azospirillum species [3, 4]

Summary

To whom correspondence should be addressed

By genetic evidence, a third pathway of xylose degradation was proposed for the bacterium Caulobacter crescentus, in analogy to an alternative catabolic pathway of L-arabinose, reported for some bacteria, including species of Azospirillum, Pseudomonas, Rhizobium, Burkholderia, and Herbasprillum [2, 3]. In these organisms L-arabinose is oxidatively degraded to ␣-ketoglutarate, an intermediate of the tricarboxylic acid cycle, via the activities of L-arabinose dehydrogenase, L-arabinolactonase, and two successive dehydration reactions forming 2-keto-3-deoxy-L-arabinoate and ␣-ketoglutarate semialdehyde; the latter compound is further oxidized to ␣-ketoglutarate via NADPϩ-specific ␣-ketoglutarate semialdehyde dehydrogenase (KGSADH).. The data show that D-xylose was exclusively degraded to ␣-ketoglutarate involving xylose dehydrogenase, a novel xylonate dehydratase, 2-keto-3-deoxyxylonate dehydratase, and ␣-ketoglutarate semialdehyde dehydrogenase

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION