Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - Supplemental Figure 1. Purification of MKS1. SDS-PAGE of crude extract and enzyme purified from E. coli. Lane 1: molecular markers; Lane 2: crude protein extract from E. coli control culture. Lane 3: crude protein extract from E. coli expressing MKS1. Lane 4: fraction with highest MKS1 concentration eluting from DE53 column. Lane 5, fraction with highest MKS1 concentration eluting from Phenyl HP column. The 13% SDS-PAGE gel was stained with Coomassie blue.
• Supplemental Figure 2 - Supplemental Figure 2. Schematic of the Synthesis of [3-¹⁴C] β-Ketomyristoyl-ACP. Synthesis started with lauric acid (C12:0) labeled at the carboxyl carbon (C1; labeled with an asterisk). The boxes below each reaction indicate the components included in the reaction. To monitor the production of [3-¹⁴C] β-ketoacyl-ACP, the intermediate product was analyzed by reduction with NaH4B followed by radio-TLC. Reduction of [3-¹⁴C] β-ketomyristoyl-ACP will result in a C₁₄; 1,3-diol, but reduction of [1-¹⁴C] 12:O acyl-ACP and [1-¹⁴C] 12:0 fatty acid will both result in a C12 1-ol. Similar procedures were conducted to synthesize [3-¹⁴C] β-ketolauroyl-ACP and [3-¹⁴C] β-ketopalmitoyl-ACP, starting with [1-¹⁴C] decanoic acid (C10:0) and [1-¹⁴C] myristic acid (C14:0), respectively. Due to the relatively low efficiency of the E. coli acyl ACP synthetase (Aas) in the acylation of decanoic acid compared to lauric acid and myristic acid (Ray and Cronan, 1976, PNAS, 73, 4374-4378.), the procedure resulted in much lower yields of the final product, [3-¹⁴C] β-ketolauroyl-ACP. The product of step I ([1-¹⁴C] Acyl-ACP) was purified on a Ni²⁺-NTA column, the product of step II ([3-¹⁴C] β-ketoacyl-ACP) was purified by TCA precipitation, and the product of step III ([2-C] 2-methylketone) was extracted with hexane (see Methods).
• Supplemental Figure 3 - Supplemental Figure 3. Radio-TLC Verification of the Production of [3-¹⁴C] β-Ketomyristoyl-ACP. Aliquots of the Step II reaction (Supplemental Figure 1) taken after 0, 2, 5 and 10 minutes were reduced with NaH4B and run on TLC. The major band at the bottom is 1,3-C₁₄ diol, which is the reduction product of [3-¹⁴C] β-ketomyristoyl-ACP. The top band is the C₁₂ 1-ol, which is derived from both free lauric acid (the substrate in step I) and lauroyl-ACP (the product of step I). Arrows indicate positions of the standards used for the identification. Similar procedures were used to verify the synthesis of [3-¹⁴C] β-ketolauroyl-ACP and [3-¹⁴C] β-ketopalmitoyl-ACP, starting with [1-¹⁴C] decanoic acid (C10:0) and [1-¹⁴C] myristic acid (C14:0), respectively.
• Supplemental Figure 4 - Supplemental Figure 4. GC-MS Analysis of the Product of the Reaction Catalyzed by MKS1 with β-Ketomyristoyl-ACP as the Substrate GC-MS of the reaction product obtained by Solid Phase MicroExtraction (SPME). The major peak (at 18.05 min) was identified as 2-tridecanone based on retention time and mass spectra (inset). GC-MS of authentic 2-tridecanone standard.
• Supplemental Figure 5 - Supplemental Figure 5. Sequence Alignment of Selected Plant Proteins Belonging to the α/β-Hydrolase (Esterase) Family.
• Supplemental Methods