A ¹⁹F‐NMR study of the membrane‐binding region of D‐lactate dehydrogenase of escherichia coli

Abstract

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 (SF-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 SF-Trp-labeled enzymes using I9F-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 I9F-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.

The crystal structure of d- lactate dehydrogenase, a peripheral membrane respiratory enzyme

“…Furthermore, residues 280 and 309 are within 15-20 Å of the membrane, which is in the range of the effect of nitroxide-spin-label on the 19 F resonance. Residue 279, postulated (13) to be close to the lipid phase, belongs to the cap domain, and, in the model below, is close to the membrane.…”

“…4. Previous studies (12)(13)(14)(15) have shown that residues Phe-339, Phe-340, Phe-341, Phe-356, Phe-357, and Phe-361, among others, are likely to be close to the lipid phase. These residues are part of the modeled region and lie within 7-12 Å of the membrane.…”

Physiological role of GlpB of anaerobic glycerol-3-phosphate dehydrogenase ofEscherichia coli

“…D-Lactate dehydrogenase interacts with the cytoplasmic membrane and requires detergent for solubilization, yet has no lengthy stretches of hydrophobic amino acids. It apparently interacts with the membrane via several separated structural elements (Sun et al 1993). We now show that the GlpAC dimer transfers its reducing equivalents to the terminal reductase via GlpB, perhaps in a manner analogous to proline dehydrogenase, although we have not been able to distinguish a difference in binding associated with redox state (M.E.R.…”

Differential effects of isomeric incorporation of fluorophenylalanines into PvuII endonuclease

“…Although there are examples of incorporation of fluorinated aliphatic amino acids,6 most fluorine atoms are incorporated into proteins as part of the aromatic side‐chains Tyr (typically as 3‐fluoro‐Tyr), Trp (4‐, 5‐, or 6‐fluoro‐Trp),7, 8 or Phe (2‐, 3‐, or 4‐fluoro‐Phe) 9. Introduction of fluorine into aromatic rings represents a very small overall change in amino acid side‐chain volume, which is quite conservative compared with typical substitutions introduced by mutagenesis 4…”

“…However, given that aromatic residues are often involved in complex interactions both in the interior of the protein and at the surface, differential effects of incorporating various fluorinated isomers at locations removed from enzyme active sites are possible. In spite of this, only a few studies involving isomeric fluorine incorporation have been reported,8, 11 and fewer still that compare the effects of these modifications on activity and conformation 9…”