Natural Sciences at Parkland College - Fall 2017

The Parkland College Natural Sciences Department Newsletter for Fall 2017 -- this issue features an article on IR cameras, use of display case, engaging students outside the classroom with the Astronomy Club and the Parkland Science Club, the solar eclipse, updates from the professional development subcommittee for faculty, summaries from events and meetings, a report on Phenotypic Pasticity Research Experience for Community College Students (PRECS) first summer, and a special feature from former professor Rich Blazier, with a special feature on the history of the Natural Sciences Department

Studies on lipoic acid metabolism

The covalent attachment of lipoic acid to the lipoyl domains (LDs) of the central metabolism enzymes pyruvate dehydrogenase (PDH) and oxoglutarate dehydrogenase (OGDH) is essential for their activation and thus for respiratory growth in the bacterium Escherichia coli and the yeast Saccharomyces cerevisiae. A third lipoic acid-dependent enzyme system, the glycine cleavage system (GCV), is required for utilization of glycine as a nitrogen source. In E. coli, lipoic acid is assembled on the LDs from the eight-carbon fatty acid, octanoate, in two steps. First, an octanoyltransferase (LipB) transfers an octanoyl moiety from the acyl carrier protein (ACP) of fatty acid biosynthesis to the LDs. A thioester-bound acyl-enzyme intermediate is formed in the process. Then lipoyl synthase (LipA) catalyzes replacement of single hydrogen atoms at carbons 6 and 8 with sulfur atoms using radical SAM chemistry. Alternatively, either exogenous lipoic acid or octanoate can be directly attached to the LDs by lipoate-protein ligase (LplA) via an acyl–AMP intermediate. E. coli strains containing null mutations in lipB are auxotrophic for either lipoic acid (or octanoate), or acetate plus succinate which respectively bypass the PDH- and OGDH-catalyzed steps required for aerobic growth on glucose minimal media. Spontaneously-arising mutant strains that retained the lipB mutation, yet did not require supplementation for aerobic growth were isolated. Initial characterization distinguished two types of suppressor strains. In chapter 2 I describe one type in which suppression was caused by single missense mutations within the coding sequence of the lplA gene. The LplA proteins encoded by the mutant genes had reduced Km values for free octanoate, which was detected in the cytoplasm at a concentration of about 28.2 μM, well above the Km values for the mutant LplA proteins. Thus in these suppressor strains, the mutant LplA proteins utilize the cytoplasmic octanoate pool to activate PDH and OGDH enabling growth. In the second type of lipB suppressor strains, the causative mutation was a stop codon in the sdhB gene, which encodes a subunit of succinate dehydrogenase (SDH). In chapter 3, these lipB sdhB strains are further characterized. I show that these strains contain active PDH and require a functional lplA gene. Succinate in this strain is produced by three enzymes, any one of which will suffice in the absence of SDH. These three enzymes are: trace levels of OGDH, the isocitrate lyase of the glyoxylate shunt, and aspartate oxidase, the enzyme catalyzing the first step of nicotinamide biosynthesis. In chapter 4, I characterize the lipoate-protein ligase of the yeast S. cerevisiae. The E. coli LplA was the first lipoate-protein ligase (Lpl) to be characterized. It catalyzes two partial reactions: activation of the acyl chain by formation of acyl-AMP, followed by transfer of the acyl chain to the LDs. It turns out that there is a surprising diversity within the Lpl family of enzymes, several of which catalyze reactions other than ligation reactions. For example, the Bacillus subtilis Lpl homologue LipM is an octanoyltransferase that transfers the octanoyl moiety from octanoyl-ACP specifically to GCV. Another B. subtilis Lpl homologue, LipL, transfers octanoate from octanoyl-GCV to other LDs in an amido-transfer reaction. In chapter 4, I report that the Lip3 Lpl homologue of S. cerevisiae has octanoyl-CoA:protein transferase activity, and discuss implications of this activity on the physiological role of Lip3 in lipoic acid synthesis

Scavenging of Cytosolic Octanoic Acid by Mutant LplA Lipoate Ligases Allows Growth of Escherichia coli Strains Lacking the LipB Octanoyltransferase of Lipoic Acid Synthesis▿

The LipB octanoyltransferase catalyzes the first step of lipoic acid synthesis in Escherichia coli, transfer of the octanoyl moiety from octanoyl-acyl carrier protein to the lipoyl domains of the E2 subunits of the 2-oxoacid dehydrogenases of aerobic metabolism. Strains containing null mutations in lipB are auxotrophic for either lipoic acid or octanoic acid. We report the isolation of two spontaneously arising mutant strains that allow growth of lipB strains on glucose minimal medium; we determined that suppression was caused by single missense mutations within the coding sequence of the gene (lplA) that encodes lipoate-protein ligase. The LplA proteins encoded by the mutant genes have reduced Km values for free octanoic acid and thus are able to scavenge cytosolic octanoic acid for octanoylation of lipoyl domains