A cDNA coding for 3-ketoacyl-acyl-carrier protein (ACP) synthase III (KAS III) from spinach (Spinacia oleracea; So KAS III) was used to isolate two closely related KAS III clones (Ch KAS III-1 and Ch KAS III-2) from Cuphea hookeriana. Both Ch KAS IIIs are expressed constitutively in all tissues examined. An increase in the levels of 16:0 was observed in tobacco (Nicotiana tabacum, WT-SR) leaves overexpressing So KAS III when under the control of the cauliflower mosaic virus-35S promoter and in Arabidopsis and rapeseed (Brassica napus) seeds overexpressing either of the Ch KAS IIIs driven by napin. These data indicate that this enzyme has a universal role in fatty acid biosynthesis, irrespective of the plant species from which it is derived or the tissue in which it is expressed. The transgenic rapeseed seeds also contained lower levels of oil as compared with the wild-type levels. In addition, the rate of lipid synthesis in transgenic rapeseed seeds was notably slower than that of the wild-type seeds. The results of the measurements of the levels of the acyl-ACP intermediates as well as any changes in levels of other fatty acid synthase enzymes suggest that malonyl-ACP, the carbon donor utilized by all the 3-ketoacyl-ACP synthases, is limiting in the transgenic plants. This further suggests that malonyl-coenzyme A is a potential limiting factor impacting the final oil content as well as further extension of 16:0.

Cocoa (Theobroma cacao L.) seeds are the source of chocolate flavor. The flavor develops upon post-harvest fermentation during which seed proteins are degraded. From 100 days after pollination (DAP) to maturity (160-180 DAP), three major protein bands (44, 26 and 21 kDa) are present in seed extracts subjected to denaturing polyacrylamide gel electrophoresis. The 44 and 26 kDa proteins, making up 30-50~o of total mature seed protein, behave as classical storage proteins [1], in contrast to the 21 kDa protein which increases during development but does not degrade to the same extent upon germination.Eleven percent of 20000 clones from a 130 DAP cocoa seed 2gtl0 library were positive when probed with synthetic oligonucleotides derived from a portion (residues 4-14) of the 21 kDa protein's N-terminal amino acid sequence (AlaAsn-Ser-Pro-Val-Leu-Asp-Thr-Asp-Gly-AspGlu-Leu-Gln-Thr-His-Val-Gln-Tyr-Tyr).The nucleotide and deduced amino acid sequences of an essentially full-length cDNA are shown in Figure 1. The transcript includes a 5' 78-nucleotide sequence for a 26-amino acid signal peptide which is not present at the N-terminus of the mature protein and a 3' 54-nucleotide poly(A) + tract, preceeded by two 3' AAUAAA elements. The calculated molecular weight of the mature protein (21331 Da)is similar to the sizes of protease inhibitors of the soybean trypsin inhibitor (Kunitz) class.The deduced amino acid sequence of the cocoa seed protein shows 38~o identity to a barely ct-amylase/subtilisin inhibitor (BASI [5], Fig. 2). The areas of greatest homology between the two proteins reflect areas of homology between them and two other Kunitz-type inhibitors (trypsin inhibitors of soybean [4] and winged bean [6], Fig. 2). Approximately 74~o (25 out of 34) of the residues conserved in all three of the protease inhibitors shown in Fig. 2 are also common to the cocoa protein. Considerably more identity is found among the sequences of the four proteins in the first 65 residues (35 of 65 residues of the cocoa protein matching any of the other three proteins) than in the middle or C-terminal thirds of the proteins. In addition, the highly conserved region between residues 4 and 24 (12 out of 24) is also highly conserved between at least four other Kunitz-type protease inhibitors from seeds of leguminous plants [3]. Four cysteine residues strictly conserved among the amino acid sequences of the protease inhibitors, and believed to be involved in disulfide bonding in BASI, are also conserved in the cocoa seed protein. The protein also shows much similarity (34~o identity) to sporamin b [2] of sweet potato tubers (Fig. 2). Many areas of high homology between the two The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X54509 Fig. 1. Nucleotide and deduced amino acid sequence of a cDNA clone encoding the 21 kDa cocoa seed protein. The arrow indicates the probable cleavage site of the putative 26-amino acid signal polypeptide from the mature protein. The u...

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Students' understandings of foundational concepts such as noncovalent interactions, pH and pK are crucial for success in undergraduate biochemistry courses. We developed a guided-inquiry activity to aid students in making connections between noncovalent interactions and pH/pK . Students explore these concepts by examining the primary and tertiary structures of immunoglobulin G (IgG) and Protein A. Students use PyMOL, an open source molecular visualization application, to (1) identify hydrogen bonds and salt bridges between and within the proteins at physiological pH and (2) apply their knowledge of pH/pK to association rate constant data for these proteins at pH 4 and pH 11. The laboratory activity was implemented within a one semester biochemistry laboratory for students majoring in allied health disciplines, engineering, and biological sciences. Several extensions for more advanced students are discussed. Students' overall performance highlighted their ability to successfully complete tasks such as labeling and identifying noncovalent interactions and revealed difficulties with analyzing noncovalent interactions under varying pH/pK conditions. Students' evaluations after completing the activity indicated they felt challenged but also recognized the potential of the activity to help them gain meaningful understanding of the connections between noncovalent interactions, pH, pK , and protein structure. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(6):528-536, 2017.