Algal Kainoid Synthases Exhibit Substrate-Dependent Hydroxylation and Cyclization Activities.

FeII/α-ketoglutarate-dependent dioxygenases (Fe/αKG) make up a large enzyme family that functionalize C-H bonds on diverse organic substrates. Although Fe/αKG homologues catalyze an array of chemically useful reactions, hydroxylation typically predominates. Microalgal DabC uniquely forms a novel C-C bond to construct the bioactive pyrrolidine ring in domoic acid biosynthesis; however, we have identified that this kainoid synthase exclusively performs a stereospecific hydroxylation reaction on its cis substrate regioisomer. Mechanistic and kinetic analyses with native and alternative substrates identified a 20-fold rate increase in DabC radical cyclization over ß-hydroxylation with no observable 1,5-hydrogen atom transfer. Moreover, this dual activity was conserved among macroalgal RadC1 and KabC homologues and provided insight into substrate recognition and reactivity trends. Investigation of this substrate-dependent chemistry improves our understanding of kainoid synthases and their biocatalytic application.

Biosynthesis of Guanitoxin Enables Global Environmental Detection in Freshwater Cyanobacteria.

Harmful cyanobacterial blooms (cyanoHABs) cause recurrent toxic events in global watersheds. Although public health agencies monitor the causal toxins of most cyanoHABs and scientists in the field continue developing precise detection and prediction tools, the potent anticholinesterase neurotoxin, guanitoxin, is not presently environmentally monitored. This is largely due to its incompatibility with widely employed analytical methods and instability in the environment, despite guanitoxin being among the most lethal cyanotoxins. Here, we describe the guanitoxin biosynthesis gene cluster and its rigorously characterized nine-step metabolic pathway from l-arginine in the cyanobacterium Sphaerospermopsis torques-reginae ITEP-024. Through environmental sequencing data sets, guanitoxin (gnt) biosynthetic genes are repeatedly detected and expressed in municipal freshwater bodies that have undergone past toxic events. Knowledge of the genetic basis of guanitoxin biosynthesis now allows for environmental, biosynthetic gene monitoring to establish the global scope of this neurotoxic organophosphate.

Homozygous Expression of Mutant ELOVL4 Leads to Seizures and Death in a Novel Animal Model of Very Long-Chain Fatty Acid Deficiency.

Lipids are essential components of the nervous system. However, the functions of very long-chain fatty acids (VLC-FA; ≥ 28 carbons) in the brain are unknown. The enzyme ELOngation of Very Long-chain fatty acids-4 (ELOVL4) catalyzes the rate-limiting step in the biosynthesis of VLC-FA (Agbaga et al., Proc Natl Acad Sci USA 105(35): 12843-12848, 2008; Logan et al., J Lipid Res 55(4): 698-708, 2014), which we identified in the brain as saturated fatty acids (VLC-SFA). Homozygous mutations in ELOVL4 cause severe neuropathology in humans (Ozaki et al., JAMA Neurol 72(7): 797-805, 2015; Mir et al., BMC Med Genet 15: 25, 2014; Cadieux-Dion et al., JAMA Neurol 71(4): 470-475, 2014; Bourassa et al., JAMA Neurol 72(8): 942-943, 2015; Aldahmesh et al., Am J Hum Genet 89(6): 745-750, 2011) and are post-natal lethal in mice (Cameron et al., Int J Biol Sci 3(2): 111-119, 2007; Li et al., Int J Biol Sci 3(2): 120-128, 2007; McMahon et al., Molecular Vision 13: 258-272, 2007; Vasireddy et al., Hum Mol Genet 16(5): 471-482, 2007) from dehydration due to loss of VLC-SFA that comprise the skin permeability barrier. Double transgenic mice with homozygous knock-in of the Stargardt-like macular dystrophy (STDG3; 797-801_AACTT) mutation of Elovl4 with skin-specific rescue of wild-type Elovl4 expression (S + Elovl4 mut/mut mice) develop seizures by P19 and die by P21. Electrophysiological analyses of hippocampal slices showed aberrant epileptogenic activity in S + Elovl4 mut/mut mice. FM1-43 dye release studies showed that synapses made by cultured hippocampal neurons from S + Elovl4 mut/mut mice exhibited accelerated synaptic release kinetics. Supplementation of VLC-SFA to cultured hippocampal neurons from mutant mice rescued defective synaptic release to wild-type rates. Together, these studies establish a critical, novel role for ELOVL4 and its VLC-SFA products in regulating synaptic release kinetics and epileptogenesis. Future studies aimed at understanding the molecular mechanisms by which VLC-SFA regulate synaptic function may provide new targets for improved seizure therapies.