Identification of a heat-stable palmitoyl/oleoyl specific acyl-acyl carrier protein thioesterase in developing seeds of Madhuca butyracea

Seed fat formation in Madhuca butyracea (also known as Bassia butyracea), a sub-Himalayan tree of Sapotaceae family, was investigated during the seed development period from 45 DAF (days after flowering) to near maturity at 93 DAF. Although the lipid content per seed kernel increased from 2.5 mg (1.85%, w/w) at 45 DAF to 525 mg (37.5% w/w) at 93 DAF, the composition of major fatty acids in the lipid did not change appreciably from the early (palmiate, 54%; oleate, 22%; stearate, 2.5% and linoleate, 13%) to the mature stage (palmitate, 66%; oleate, 27.5%: stearate, 2.5% and linoleate, 3%), indicating high content of palmitate in its seed fat (local name Phulwara fat). To investigate the possible role of any palmitoyl specific fatty acyl-ACP thioesterase (FAT) that could be responsible for the accumulation of this unique high-palmitate fat in its seed, FAT activities in the extracts of developing seed kernels (as well as that of young leaves and flow er buds) were analyzed. In addition to housekeeping oleoyl preferring LC-FAT (apparent pI, 8.2). a novel FAT showing almost equal substrate specificity for palmitoyl-and oleoyl-ACP. which we termed as PO-FAT, was found at high levels in seed kernels and to a smaller extent in leaves and flower buds. The enzyme (PO-FAT) was purified and biochemically characterized. The relative rate of substrate (at 2 muM) hydrolysis by the purified enzyme was 100:90:30:7 for palmitoyl-, oleoyl-, stearoyl- and lauroyl-ACP, respectively, and the V-max/K-m ratios for the first three substrates were 100:73:47. respectively. The enzyme was found to be a heat-stable basic protein (apparent pI, 9.7) showing its optimum activity at pH 9.0. It is highly probable that the enzyme PO-FAT plays a pivotal role in determining the fatty acid composition of the palmitate-rich Phulwara fat in developing seeds of the plant. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.