Question

Approximately 4% of cellular enzymes use coenzyme A. Understanding the role of this cofactor is critical to understanding many metabolic processes, such as glucose oxidation and fatty acid oxidation.

A) What is coenyme A and what are its important structural features?

B) What is its role (or the role of acetyl-CoA) in metabolism?

C) What advantages does this cofactor impart?

Answer 1

Coenzyme A (CoA, CoASH, or HSCoA) is a coenzyme, notable for its role in the synthesis and oxidation of fatty acids, and the oxidation of pyruvate in the citric acid cycle. All genomes sequenced to date encode enzymes that use coenzyme A as a substrate, and around 4% of cellular enzymes use it (or a thioester, such as acetyl-CoA) as a substrate. In humans, CoA biosynthesis requires cysteine, pantothenate, and adenosine triphosphate .

Since coenzyme A is, in chemical terms, a thiol, it can react with carboxylic acids to form thioesters, thus functioning as an acyl group carrier. It assists in transferring fatty acidsfrom the cytoplasm to mitochondria. A molecule of coenzyme A carrying an acetyl group is also referred to as acetyl-CoA. When it is not attached to an acyl group, it is usually referred to as 'CoASH' or 'HSCoA'.

Coenzyme A is also the source of the phosphopantetheine group that is added as a prosthetic group to proteins such as acyl carrier protein and formyltetrahydrofolate dehydrogenase.^[4][5] The portion of Coenzyme A that reacts with potential substrates is the thiol group.

B Acetyl coenzyme A (acetyl-CoA) is the central intermediate of the pathways required to metabolize nonfermentable carbon sources. Three such pathways, i.e., gluconeogenesis, the glyoxylate cycle, and beta-oxidation, are required for full virulence in the fungal pathogen Candida albicans. These processes are compartmentalized in the cytosol, mitochondria, and peroxosomes, necessitating transport of intermediates across intracellular membranes. Acetyl-CoA is trafficked in the form of acetate by the carnitine shuttle, and we hypothesized that the enzymes that convert acetyl-CoA to/from acetate, i.e., acetyl-CoA hydrolase (ACH1) and acetyl-CoA synthetase (ACS1 and ACS2), would regulate alternative carbon utilization and virulence. We show that C. albicans strains depleted for ACS2 are unviable in the presence of most carbon sources, including glucose, acetate, and ethanol; these strains metabolize only fatty acids and glycerol, a substantially more severe phenotype than that of Saccharomyces cerevisiae acs2 mutants. In contrast, deletion of ACS1 confers no phenotype, though it is highly induced in the presence of fatty acids, perhaps explaining why acs2 mutants can utilize fatty acids. Strains lacking ACH1 have a mild growth defect on some carbon sources but are fully virulent in a mouse model of disseminated candidiasis. Both ACH1 and ACS2 complement mutations in their S. cerevisiae homolog. Together, these results show that acetyl-CoA metabolism and transport are critical for growth of C. albicans on a wide variety of nutrients. Furthermore, the phenotypic differences between mutations in these highly conserved genes in S. cerevisiae and C. albicans support recent findings that significant functional divergence exists even in fundamental metabolic pathways between these related yeasts.

At this point, it should be pretty clear that the number of options when designing an expression system is considerably high. Choosing the perfect combination is not possible a priori, so multiple conditions should be tested to obtain the desired protein. If the project demands expressing two protein constructs, cloned in six different expression vectors, each transformed in three different expression strains, then you are in for 36 expression trials. This number may be even higher when other variables are taken into account. This trial-and-error and time consuming pilot study can be made faster if micro-expression trials are performed before scale-up. Small-scale screens can be performed in 2-ml tubes or 96-well plates (Shih et al., 2002). High throughput protocols adapting automatic liquid handling robots have been described, making it possible for a single person to test more than 1000 culture conditions within a week.