A 19F-NMR study of the membrane-binding region of D-lactate dehydrogenase of Escherichia coli.
D-Lactate dehydrogenase (D-LDH) is a membrane-associated respiratory enzyme of Escherichia coli. The protein is composed of 571 amino acid residues with a flavin adenine dinucleotide (FAD) cofactor, has a molecular weight of approximately 65,000, and requires lipids or detergents for full activity. We used NMR spectroscopy to investigate the structure of D-LDH and its interaction with phospholipids. We incorporated 5-fluorotryptophan (5F-Trp) into the native enzyme, which contains five tryptophan residues, and into mutant enzymes, where a sixth tryptophan is substituted into a specific site by oligonucleotide-directed mutagenesis, and studied the 5F-Trp-labeled enzymes using 19F-NMR spectroscopy. In this way, information was obtained about the local environment at each native and substituted tryptophan site. Using a nitroxide spin-labeled fatty acid, which broadens the resonance from any residue within 15 A, we have established that the membrane-binding area of the protein includes the region between Tyr 228 and Phe 369, but is not continuous within this region. This conclusion is strengthened by the results of 19F-NMR spectroscopy of wild-type enzyme labeled with fluorotyrosine or fluorophenylalanine in the presence and absence of a nitroxide spin-labeled fatty acid. These experiments indicate that 9-10 Phe and 3-4 Tyr residues are located near the lipid phase.
Sun
Z Y
ZY
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213.
Truong
H T
HT
Pratt
E A
EA
Sutherland
D C
DC
Kulig
C E
CE
Homer
R J
RJ
Groetsch
S M
SM
Hsue
P Y
PY
Ho
C
C
D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005461:Fluorine / Q000737:chemistry; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D045022:Lactate Dehydrogenases; D009682:Magnetic Resonance Spectroscopy; D008567:Membranes, Artificial; D016297:Mutagenesis, Site-Directed; D013113:Spin Labels; D014364:Tryptophan / Q000031:analogs & derivatives / Q000737:chemistry; D014443:Tyrosine / Q000031:analogs & derivatives / Q000737:chemistry
D008567:Membranes, Artificial; D013113:Spin Labels; C001245:3-fluorotyrosine; D005461:Fluorine; C026927:5-fluorotryptophan; D014443:Tyrosine; D014364:Tryptophan; D045022:Lactate Dehydrogenases; D007770:L-Lactate Dehydrogenase; C071195:D-lactate dehydrogenase