Recombinant Prochlorococcus marinus 2-dehydro-3-deoxyphosphooctonate aldolase (kdsA)

Introduction to Prochlorococcus marinus and KdsA

Prochlorococcus marinus is the most abundant photosynthetic organism in the ocean and a major contributor to global carbon fixation . These tiny, emerald-colored microbes are found in vast numbers throughout the ocean's surface waters . They play a vital role in carbon fixation, contributing as much as all crops on land combined .

2-dehydro-3-deoxyphosphooctonate aldolase (KdsA) is an enzyme involved in the biosynthesis of KDO (3-deoxy-D-manno-oct-2-ulosonic acid), a sugar found in the lipopolysaccharide of gram-negative bacteria and essential for their cell wall integrity . The enzyme catalyzes the condensation of phosphoenolpyruvate and D-erythrose 4-phosphate to form KDO-8-phosphate. KdsA is an essential enzyme in bacteria, and its inhibitors have antibacterial activity .

Role of DNA Ligases in Prochlorococcus marinus

Prochlorococcus marinus encodes multiple ATP-dependent DNA ligases (AD-ligases) in their genomes, but lacks the other subunits associated with known AD-ligase-dependent repair pathways . Comparative genomics of sequenced isolates of P. marinus indicates a role for these AD-ligases in DNA replication . P. marinus ecotypes are broadly classified as high-light, which reside in the UV-damaging nutrient-poor upper ocean, and low-light, which experience less UV and have higher nutrient access . Low-light strains have the smallest genomes of any free-living organisms (1.66 -1.75 MB), yet they encode up to three predicted AD-ligases with different domain compositions .

DNA Ligase Activity and Genome Evolution

In high-light strains of P. marinus, the genes for the 'essential' replicative ND-ligases are severely affected, with the replicative recombinase RecA . AD-Lig P is active on singly nicked or cohesive breaks and prefers $$Mg^{2+}$$ as a divalent cation . AD-Lig P is more than 10 fold as effective at sealing singly-nicked double-stranded DNA substrates relative to the low-light ND-Lig fl under equivalent conditions . Genome rearrangements have played a key role in the evolution of P. marinus ecotypes .

Key Findings on DNA Ligases in High-Light Strains

All high-light strains of P. marinus lack a gene encoding a complete ND-ligase gene, meaning this essential role must be fulfilled by one of its AD-ligases . The AD-ligase AD-Lig P is substituted in the equivalent genetic context of the ND-ligase, suggesting this isoform is responsible for replicative ligase activity . The high-light AD-Lig P is an ATP-dependent DNA ligase with >10x higher specific activity relative to the low-light ND-ligase .

Antibacterial Activity

Indolylquinazolinone 3k showed high activity against staphylococci . Compound 3k inhibited the growth of S. aureus ATCC 25923, showing an MIC of 3.90 μg/mL . Its MIC against the reference strain S. aureus ATCC 43300 (MRSA) was even lower and did not exceed 1 μg/mL, which is promising because MRSA is now widespread and a significant problem in human and veterinary medicine, because it is resistant to many antibacterial compounds .

Additional Metabolic Features

Prochlorococcus marinus MED4 features a complete Entner–Doudoroff pathway, rewired glycogen metabolism, and increased coverage of the genome . To simulate the natural environmental constraints experienced by Prochlorococcus, Push-FBA is used, where light and bicarbonate uptake are given a fixed flux independent of growth rate . Glycogen metabolism in iSO595 was rewired to study the dynamic allocation of glycogen . Dynamic light conditions and light absorption in COMETS simulate the growth of P. marinus during the diel cycle . iSO595 has increased coverage both in terms of genes, reactions, and metabolites compared to its ancestor iJC568 .