Purification and characterization of 9-hexadecenoic acid cis-trans isomerase from Pseudomonas sp. strain E-3

Abstract

A 9-hexadecenoic acid cis-trans isomerase (9-isomerase) that catalyzed the cis-to-trans isomerization of the double bond of free 9-cis-hexadecenoic acid [16:1(9c)] was purified to homogeneity from an extract of Pseudomonas sp. strain E-3 and characterized. Electrophoresis of the purified enzyme on both incompletely denaturing and denaturing polyacrylamide gels yielded a single band of a protein with a molecular mass of 80 kDa, suggesting that the isomerase is a monomeric protein of 80 kDa. The 9-isomerase, assayed with 16:1(9c) as a substrate, had a specific activity of 22.8 &mgr;mol h-1 (mg protein)-1 and a Km of 117.6 mM. The optimal pH and temperature for catalysis were approximately pH 7-8 and 30 degrees C, respectively. The 9-isomerase catalyzed the cis-to-trans conversion of a double bond at positions 9, 10, or 11, but not that of a double bond at position 6 or 7 of cis-mono-unsaturated fatty acids with carbon chain lengths of 14, 15, 16, and 17. Octadecenoic acids with a double bond at position 9 or 11 were not susceptible to isomerization. These results suggest that 9-isomerase has a strict specificity for both the position of the double bond and the chain length of the fatty acid. The enzyme catalyzed the cis-to-trans isomerization of fatty acids in a free form, and in the presence of a membrane fraction it was also able to isomerize 16:1(9c) esterified to phosphatidylethanolamine. The 9-isomerase was strongly inhibited by catecholic antioxidants such as alpha-tocopherol and nordihydroguaiaretic acid, but was not inhibited by 1, 10-phenanthroline or EDTA or under anoxic conditions. Based on these results, the possible mechanism of catalysis by this enzyme is discussed.

Formation of trans Fatty Acids Is Not Involved in Growth-Linked Membrane Adaptation of Pseudomonas putida

“…The enzyme has been purified (49,50), and the encoding gene was cloned and sequenced (24,27). It has been verified that the isomerization reaction is possible without growth (22,36).…”

Pseudomonas putidaNCTC 10936 Balances Membrane Fluidity in Response to Physical and Chemical Stress by Changing the Saturation Degree and theTrans/cisRatio of Fatty Acids

“…16,23,24) However, in growing cells of Pseudomonas strains 16,23) both changes of the saturation degree of the fatty acids by de novo synthesis of saturated and unsaturated fatty acids via the anaerobic pathway 25,26) and conversion of cis into trans fatty acids by a cis-trans isomerase 27) can occur in parallel. Therefore the elucidation of their contribution to homeoviscousis is difficult.…”

Mechanism of cis-trans Isomerization of Unsaturated Fatty Acids in Pseudomonas putida

“…The enzyme has been purified from the periplasmic fraction of Pseudomonas oleovorans (17) and Pseudomonas sp. strain E3 (16) and has been isolated as a His-tagged P. putida P8 protein heterologously expressed in Escherichia coli (9). The cis-trans isomerase gene cloned and sequenced from P. putida P8 (10,21) and P. putida DOT-T1E (12) made evident that the isomerase has an N-terminal hydrophobic signal sequence, which is cleaved off after the enzyme has been targeted to the periplasmic space.…”

“…For an enzyme preparation from Pseudomonas sp. strain E3, which is presumably homologous to the cti gene product of P. putida P8, it was suggested that iron (probably Fe 3ϩ ) plays a crucial role in the catalytic reaction (16). Despite the wealth of available information, the biochemical mechanism of the solvent-induced regulation of isomerase activity, in relation to membrane homeostasis (18), still remains obscure.…”

“…Peak numbers and retention times for the GC are indicated (see Fig. 1 16.037 min, m/z 298, doubly deuterated) were detected. The occurrence of only one peak of elaidic acid with the molecular mass m/z 298 is most interesting, as it proves that the oleic acid was exclusively converted into the doubly deuterated derivative of elaidic acid.…”