Site-directed mutants of Escherichia coli alpha-ketoglutarate permease (KgtP).
To investigate an active site(s) in the Escherichia coli alpha-ketoglutarate premease, 11 point mutants were made in the corresponding structural gene, kgtP, by oligonucleotide-directed mutagenesis and the polymerase chain reaction. On the basis of sequences conserved in KgtP and related members of a transporter superfamily [Henderson P. J. F., & Maiden, M. C. (1990) Philos. Trans. R. Soc. London B 326, 391], Arg76 was replaced with Ala, Asp, or Lys; Asp88 with Asn or Glu; His90 with Ala; Arg92 with Ala or Lys; and Arg198 with Ala, Asp, or Lys. Mutant proteins expressed using the T7 polymerase system were in each case shown to be membrane-associated. However, they differed in transport activity. Mutants H90A and R198K had activities similar to that of wild type, and R76K and R198A retained 10-60% of the wild-type activity. In all other mutants, alpha-ketoglutarate transport was abolished. The results suggest that Arg92, which is highly conserved among other members of the transporter superfamily, is necessary for activity and also that Asp88 is critical for function, as observed for the tetracycline transporter. These data show further that a positive charge is essential at position 76 and is also important, but not absolutely required, at position 198 for alpha-ketoglutarate transport. Unlike lacY permease which was inactivated by deleting the last helix [McKenna, E., Hardy, D., Pastore, J. C., & Kaback, H. R. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2969], a KgtP truncation mutant missing the last putative membrane-spanning region was relatively stable and also retained 10-50% of the wild-type level of alpha-ketoglutarate transport activity.
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Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey 08854-5638.
Shatkin
A J
AJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D029382:Dicarboxylic Acid Transporters; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D007656:Ketoglutaric Acids / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D013329:Structure-Activity Relationship; D014169:Transformation, Bacterial
D002352:Carrier Proteins; D029382:Dicarboxylic Acid Transporters; D029968:Escherichia coli Proteins; D007656:Ketoglutaric Acids; D026901:Membrane Transport Proteins; C068628:KgtP protein, E coli