A single V317A or V317M substitution in Enzyme II of a newly identified beta-glucoside phosphotransferase and utilization system of Corynebacterium glutamicum R extends its specificity towards cellobiose.
A catabolic system involved in the utilization of beta-glucosides in Corynebacterium glutamicum R and its spontaneous mutant variants allowing uptake of cellobiose were investigated. The system comprises a beta-glucoside-specific Enzyme IIBCA component (gene bglF) of the phosphotransferase system (PTS), a phospho-beta-glucosidase (bglA) and an antiterminator protein (bglG) from the BglG/SacY family of transcription regulators. The results suggest that transcription antitermination is involved in control of induction and carbon catabolite repression of bgl genes, which presumably form an operon. Functional analysis of the bglF and bglA products revealed that they are simultaneously required for uptake, phosphorylation and breakdown of methyl beta-glucoside, salicin and arbutin. Although cellobiose is not normally a substrate for BglF permease and is not utilized by C. glutamicum R, cellobiose-utilizing mutants can be obtained. The mutation responsible was mapped to the bgl locus and sequenced, and point mutations were found in codon 317 of bglF. These led to substitutions V317A and/or V317M near the putative PTS active-site H313 in the membrane-spanning IIC domain of BglF and allowed BglF to act on cellobiose. Such results strengthen the evidence that the IIC domains can be regarded as selectivity filters of the PTS.
Kotrba
Pavel
P
Research Institute of Innovative Technology for the Earth, 9-2 Kizugawadai, Kizu, Soraku, Kyoto 619-0292, Japan.
Inui
Masayuki
M
Research Institute of Innovative Technology for the Earth, 9-2 Kizugawadai, Kizu, Soraku, Kyoto 619-0292, Japan.
Yukawa
Hideaki
H
Research Institute of Innovative Technology for the Earth, 9-2 Kizugawadai, Kizu, Soraku, Kyoto 619-0292, Japan.
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D050260:Carbohydrate Metabolism; D020134:Catalytic Domain / Q000235:genetics; D002475:Cellobiose / Q000378:metabolism*; D003001:Cloning, Molecular; D003352:Corynebacterium / Q000201:enzymology* / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017354:Point Mutation; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D004269:DNA, Bacterial; D002475:Cellobiose; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System