Chimeragenesis of the fatty acid binding site of cytochrome P450BM3. Replacement of residues 73-84 with the homologous residues from the insect cytochrome P450 CYP4C7.

A protein fragment of P450BM3 (residues 73-84) which participates in palmitoleate binding was subjected to scanning chimeragenesis. Amino acids 73-84, 73-78, 75-80, and 78-82 were replaced with the homologous fragments of the insect terpenoid hydroxylase CYP4C7. The four chimeric proteins, C(73-84), C(73-78), C(75-80), and C(78-82), were expressed, purified, and characterized. All the chimeric proteins contained all the cofactors and catalyzed monooxygenation of palmitate and of the sesquiterpene farnesol. Chimeragenesis altered substrate binding as shown by the changes in the amplitude of the palmitate-induced type I spectral shift. C(78-82) had monooxygenase activities close to those of P450BM3, while the rest of the chimeric proteins had monooxygenase activities that were inhibited relative to that of wild-type P450BM3. The extent of inhibition of the chimeric proteins varied depending on the substrate, and in the case of C(73-84), farnesol and palmitate oxidation was inhibited by 1 and 4 orders of magnitude, respectively. (1)H NMR spectroscopy and GC-MS were used to identify products of farnesol and palmitate oxidation. Wild-type P450BM3 and all chimeric proteins catalyzed oxidation of farnesol with formation of 9-hydroxyfarnesol and farnesol 10,11- and 2,3-epoxides. Three of the four chimeric proteins also formed a new compound, 5-hydroxyfarnesol, which was the major product in the case of C(73-78). In addition to hydroxylation of the C13-C15 atoms, the chimeric enzymes catalyze significant hydroxylation of the C10-C12 atoms of palmitate. In the case of C(78-82), the rates of formation of 11- and 12-hydroxypalmitates increased 7-fold compared to that of wild-type P450BM3 to 106 and 212 min(-)(1), respectively, while the rate of 10-hydroxypalmitate synthesis increased from zero to 106 min(-)(1). Thus, chimeragenesis of the region of residues 73-84 of the substrate binding site shifted the regiospecificity of substrate oxidation toward the center of the farnesol and palmitate molecules.

Structure and characterization of flavolipids, a novel class of biosurfactants produced by Flavobacterium sp. strain MTN11.

Herein we report the structure and selected properties of a new class of biosurfactants that we have named the flavolipids. The flavolipids exhibit a unique polar moiety that features citric acid and two cadaverine molecules. Flavolipids were produced by a soil isolate, Flavobacterium sp. strain MTN11 (accession number AY162137), during growth in mineral salts medium, with 2% glucose as the sole carbon and energy source. MTN11 produced a mixture of at least 37 flavolipids ranging from 584 to 686 in molecular weight (MW). The structure of the major component (23%; MW = 668) was determined to be 4-[[5-(7-methyl-(E)-2-octenoylhydroxyamino)pentyl]amino]-2-[2-[[5-(7-methyl-(E)-2-octenoylhydroxyamino)pentyl]amino]-2-oxoethyl]-2-hydroxy-4-oxobutanoic acid. The partially purified flavolipid mixture isolated from strain MTN11 exhibited a critical micelle concentration of 300 mg/liter and reduced surface tension to 26.0 mN/m, indicating strong surfactant activity. The flavolipid mixture was a strong and stable emulsifier even at concentrations as low as 19 mg/liter. It was also an effective solubilizing agent, and in a biodegradation study, it enhanced hexadecane mineralization by two isolates, MTN11 (100-fold) and Pseudomonas aeruginosa ATCC 9027 (2.5-fold), over an 8-day period. The flavolipid-cadmium stability constant was measured to be 3.61, which is comparable to that for organic ligands such as oxalic acid and acetic acid. In summary, the flavolipids represent a new class of biosurfactants that have potential for use in a variety of biotechnological and industrial applications.