The D-xylose-binding protein, XylF, from Thermoanaerobacter ethanolicus 39E: cloning, molecular analysis, and expression of the structural gene.
Immediately downstream from the Thermoanaerobacter ethanolicus xylAB operon, comprising genes that encode D-xylose isomerase and D-xylulose kinase, lies a 1,101-bp open reading frame that exhibits 61% amino acid sequence identity to the Escherichia coli D-xylose binding periplasmic receptor, XylF, a component of the high-affinity binding-protein-dependent D-xylose transport. The 25-residue N-terminal fragment of the deduced T. ethanolicus XylF has typical features of bacterial leader peptides. The C-terminal portion of this leader sequence matches the cleavage consensus for lipoproteins and is followed by a 22-residue putative linker sequence rich in serine, threonine, and asparagine. The putative mature 341-amino-acid-residue XylF (calculated molecular mass of 37,069 Da) appears to be a lipoprotein attached to the cell membrane via a lipid anchor covalently linked to the N-terminal cysteine, as demonstrated by metabolic labelling of the recombinant XylF with [14C]palmitate. The induced E. coli avidly bound D-[14C]xylose, yielding additional evidence that T. ethanolicus XylF is the D-xylose-binding protein. On the basis of sequence comparison of XylFs to other monosaccharide-binding proteins, we propose that the sequence signature of binding proteins specific for hexoses and pentoses be refined as (KDQ)(LIVFAG)3IX3(DN)(SGP)X3(GS)X(LIVA) 2X2A. Transcription of the monocistronic 1.3-kb xylF mRNA is inducible by xylose and unaffected by glucose. Primer extension analysis indicated that xylF transcription initiates from two +1 sites, both situated within the xylAB operon. Unlike in similar transport systems in other bacteria, the genes specifying the membrane components (e.g., ATP-binding protein and permease) of the high-affinity D-xylose uptake system are not located in the vicinity of xylF in T. ethanolicus. This is the first report of a gene encoding a xylose-binding protein in a gram-positive or thermophilic bacterium.
Erbeznik
M
M
Department of Animal Sciences, University of Kentucky, Lexington, Kentucky 40546-0215, USA.
Strobel
H J
HJ
Dawson
K A
KA
Jones
C R
CR
D000595:Amino Acid Sequence; D001421:Bacteria, Anaerobic / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D029968:Escherichia coli Proteins; D016988:Gram-Positive Asporogenous Rods, Irregular / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D027981:Symporters; D014994:Xylose / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D027981:Symporters; C033903:xylE protein, E coli; D014994:Xylose