EVIDENCE THAT HIS(110) OF THE PROTEIN FADL IN THE OUTER-MEMBRANE OF ESCHERICHIA-COLI IS INVOLVED IN THE BINDING AND UPTAKE OF LONG-CHAIN FATTY-ACIDS - POSSIBLE ROLE OF THIS RESIDUE IN CARBOXYLATE BINDING
Black, PN; Zhang, Q
| HERO ID | 4936719 |
|---|---|
| In Press | No |
| Year | 1995 |
| Title | EVIDENCE THAT HIS(110) OF THE PROTEIN FADL IN THE OUTER-MEMBRANE OF ESCHERICHIA-COLI IS INVOLVED IN THE BINDING AND UPTAKE OF LONG-CHAIN FATTY-ACIDS - POSSIBLE ROLE OF THIS RESIDUE IN CARBOXYLATE BINDING |
| Authors | Black, PN; Zhang, Q |
| Journal | Biochemical Journal |
| Volume | 310 |
| Page Numbers | 389-394 |
| Abstract | The binding of exogenous fatty acids to the outer-membrane protein FadL of Escherichia coli is specific for long-chain fatty acids (C-14-C-18). Oleoyl alcohol [(Z)-9-octadecen-1-o1] and methyl oleate were unable to displace FadL-specific binding of [H-3]oleate (C-18:1), suggesting that the carboxylate of the long-chain fatty acid was required for binding. Therefore the binding of exogenous fatty acids to FadL is governed, in part, by the carboxy group of the long-chain fatty acid. Treatment of whole cells with I mM diethyl pyrocarbonate (DEPC) depressed binding by 43-73 % over the range of oleate concentrations used (10-500 nM). On the basis of these results and the notion that histidine residues often play a role involving proton transfer and charge-pairing, the five histidine residues within FadL (His(110), His(226), His(327), His(345) and His(418)) were replaced by alanine using site-directed mutagenesis. Altered FadL proteins were correctly localized in the outer membrane at wild-type levels and retained the heat-modifiable property characteristic of the wild-type protein. Initial screening of these fadL mutants revealed that the replacement of His(110) by Ala resulted in a decreased growth rate on minimal oleate/agar plates. The rates of long-chain fatty acid transport for Delta fadL strains harbouring each mutation on a plasmid, with the exception of fadLH110A, were the same, or nearly the same, as those for the wild-type. fadLH110A was also defective in binding, arguing that the functional effect of this mutation was at the level of long-chain-fatty-acid binding. Other mutants had levels of long-chain-fatty-acid binding that were either the same, or nearly the same, as those for the wild-type. On the basis of competition experiments, DEPC treatment and the analyses of the mutants, His(110) may function in carboxylate binding. |
| Doi | 10.1042/bj3100389 |
| Wosid | WOS:A1995RR74300006 |
| Is Certified Translation | No |
| Dupe Override | 4936719 |
| Is Public | Yes |