We have purified membrane-bound fatty acid (omega-1-omega-3) hydroxylase of the fungus Fusarium oxysporum MT-811 and found that the activity depends on a single polypeptide with an apparent M(r) value of 118,000. The purified hydroxylase exhibited spectral characteristics of cytochrome P450 (P450), and could catalyze the hydroxylation without the aid of any other proteinaceous components, such as NADPH-P450 reductase. These properties of the fungal hydroxylase are the same as those of bacterial P450BM3 of Bacillus megaterium, a catalytically self-sufficient fused protein of P450 and its reductase. Other properties of the two enzymes, such as molecular weight, high catalytic turnover, and the regiospecificity of the hydroxylating position, were also almost identical. Further, the fungal hydroxylase reacted with the antibody to P450BM3. It was thus shown that the fungal fatty acid hydroxylase reacted with the antibody to P450BM3. It was thus shown that the fungal fatty acid hydroxylase structurally and functionally bears a close resemblance to P450BM3, although it is membrane-bound, unlike the bacterial counterpart. On the other hand, a unique phenomenon was found with the fungal hydroxylase: its NADPH-cytochrome c- or NADPH-menadione reductase activity was enhanced enormously upon binding of its substrate (fatty acid). This appears to be the first instance in which the reactivity of P450 reductase against an artificial electron acceptor was enhanced by the binding of the substrate (to be hydroxylated) to P450. These results raise interesting questions about the molecular evolution of P450. Here we term the fungal hydroxylase cytochrome P450foxy.

The plasma membrane acts as the primary interface between the cellular cytoplasm and the extracellular environment. To investigate the function of the plasma membrane in response to flooding stress, plasma membrane was purified from root and hypocotyl of soybean seedlings using an aqueous two-phase partitioning method. Purified plasma membrane proteins with 81% purity were analyzed using either two-dimensional polyacrylamide gel electrophoresis followed by mass spectrometry and protein sequencing (2-DE MS/sequencer)-based proteomics or nanoliquid chromatography followed by mass spectrometry (nanoLC-MS/MS)-based proteomics. The number of hydrophobic proteins identified by nanoLC-MS/MS-based proteomics was compared with those identified by 2-DE MS/sequencer-based proteomics. These techniques were applied to identify the proteins in soybean that are responsive to flooding stress. Results indicate insights of plasma membrane into the response of soybean to flooding stress: (i) the proteins located in the cell wall are up-regulated in plasma membrane; (ii) the proteins related to antioxidative system play a crucial role in protecting cells from oxidative damage; (iii) the heat shock cognate protein plays a role in protecting proteins from denaturation and degradation during flooding stress; and (iv) the signaling related proteins might regulate ion homeostasis.