Site-specific incorporation of 5-fluorotryptophan as a probe of the structure and function of the membrane-bound D-lactate dehydrogenase of Escherichia coli: a 19F nuclear magnetic resonance study.
The structure and function of the membrane-bound D-lactate dehydrogenase of Escherichia coli have been investigated by fluorine-19 nuclear magnetic resonance spectroscopy of 5-fluorotryptophan-labeled enzyme in conjunction with oligonucleotide-directed, site-specific mutagenesis. 5-Fluorotryptophan has been substituted for nine phenylalanine, tyrosine, and leucine residues in the enzyme molecule without loss of activity. The 19F signals from these additional tryptophan residues have been used as markers for sensitivity to substrate, exposure to aqueous solvent, and proximity to a lipid-bound spin-label. The nuclear magnetic resonance data show that two mutational sites, at amino acid residues 340 and 361, are near the lipid environment used to stabilize the enzyme. There are a number of amino acid residues on the carboxyl side of this region that are strongly sensitive to the aqueous solvent. The environment of the wild-type tryptophan residue at position 469 changes as a result of two of the substitution mutations, suggesting some amino acid residue-residue interactions. Secondary structure prediction methods indicate a possible binding site for the flavin adenine dinucleotide cofactor in the carboxyl end of the enzyme molecule. These results suggest that the membrane-bound D-lactate dehydrogenase may have the two-domain structure of many cytoplasmic dehydrogenases but with the addition of a membrane-binding domain between the catalytic and cofactor-binding domains. This type of three-domain structure may be of general significance for understanding the structure of membrane-bound proteins which do not traverse the lipid bilayer of membranes.
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213.
O B
OB
Peersen
E A
EA
Pratt
H T
HT
Truong
C
C
Ho
G S
GS
Rule
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005456:Fluorescent Dyes; D015870:Gene Expression; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D009154:Mutation; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000031:analogs & derivatives*
D005456:Fluorescent Dyes; C026927:5-fluorotryptophan; D014364:Tryptophan; D007770:L-Lactate Dehydrogenase