A novel structural basis for membrane association of a protein: construction of a chimeric soluble mutant of (S)-mandelate dehydrogenase from Pseudomonas putida.
The (S)-mandelate dehydrogenase (MDH) from Pseudomonas putida (ATCC 12633) is the only membrane-associated member of a homologous family of FMN-dependent, alpha-hydroxy acid dehydrogenases/oxidases that includes the structurally characterized glycolate oxidase from spinach (GOX). We have correlated the membrane association of MDH to a polypeptide segment in the interior of the primary sequence. This has been accomplished by construction of a chimeric enzyme in which the putative membrane-binding segment in MDH has been deleted and replaced with the corresponding segment from the soluble GOX. The resulting chimera, MDH-GOX, is soluble and retains partial catalytic activity (approximately 1%) using (S)-mandelate as substrate. In contrast, the activities of both the membrane-associated wild-type MDH and the soluble MDH-GOX are nearly the same when (S)-phenyllactate is used as substrate. To the best of our knowledge, this is the first example of a membrane-associated protein in which an internal polypeptide segment anchors the protein to the membrane.
Mitra
B
B
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742.
Gerlt
J A
JA
Babbitt
P C
PC
Koo
C W
CW
Kenyon
G L
GL
Joseph
D
D
Petsko
G A
GA
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D001483:Base Sequence; D002451:Cell Compartmentation; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000737:chemistry; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D016958:Pseudomonas putida / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000648:ultrastructure; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013057:Spectrum Analysis; D013379:Substrate Specificity
D001426:Bacterial Proteins; D017931:DNA Primers; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D000429:Alcohol Oxidoreductases; C020821:glycollate oxidase; C021538:L-mandelate dehydrogenase