We have developed hybrid P450 BM3 enzymes consisting of a Ru(II)-diimine photosensitizer covalently attached to non-native single cysteine residues of P450 BM3 heme domain mutants. These enzymes are capable, upon light activation, of selectively hydroxylating lauric acid with 40 times higher total turnover numbers compared to the peroxide shunt.

We have developed a series of hybrid P450 BM3 enzymes to perform the light-activated hydroxylation of lauric acid. These enzymes contain a Ru(II)-diimine photosensitizer covalently attached to single cysteine residues of mutant P450 BM3 heme domains. The library of hybrid enzymes includes four non-native single cysteine mutants (K97C, Q397C, Q109C and L407C). In addition, mutations around the heme active site, F87A and I401P, were inserted in the Q397C mutant. Two heteroleptic Ru(II) complexes, Ru(bpy)2phenA (1) and Ru(phen)2phenA (2) (bpy=bipyridine, phen=1,10-phenanthroline, and phenA=5-acetamido-1,10-phenanthroline), are used as photosensitizers. Upon visible light irradiation, the hybrid enzymes display various total turnover numbers in the hydroxylation of lauric acid, up to 140 for the L407C-1 mutant, a 16-fold increase compared to the F87A/Q397C-1 mutant. CO binding studies confirm the ability of the photogenerated Ru(I) compound to reduce the fraction of ferric high spin species present in the mutants upon substrate binding.

Lung cancer is the leading cause of cancer death. Non‐small cell lung cancer (NSCLC) makes up 85% of all types of lung cancer, and includes squamous cancer, adenocarcinomas, and large cell cancer. Malate dehydrogenase (MDH) exists as two forms, MDH1 (cytosolic) and MDH2 (mitochondrial). Both forms are elevated in tumor cells, but only high expression of MDH1 is associated with poor prognosis. Patients with amplified MDH1 have a 50% reduction rate of survival. MDH1 appears to support proliferation of tumor cells by fueling glycolysis with the regeneration of nicotinamide adenine dinucleotide (NAD+). However, studies of the kinetics of human MDH1 is limited. Our goal is to characterize the catalytic features and regulation of MDH1. We hypothesized that MDH1 activity is inhibited in the presence of reactive oxygen species (ROS). Human MDH1 was expressed heterogenously from Escherichia coli and purified using immobilized cobalt affinity chromatography. The enzyme catalytic activity was determined in the optimal conditions of salt, pH, temperature, and buffer. In the assay, MDH1 activity was calculated from the oxidation rate of NADH to NAD+ at 340 nm using a spectrophotometer after incubating MDH1 with oxaloacetate and NADH under steady‐state conditions. To test our hypothesis under oxidative environment, we repeated this assay using oxidized glutathione. The enzyme activity was 6‐fold lower than that measured in reducing conditions. In the absence of the enzyme, oxidized glutathione does not convert NADH to NAD+, which implied the enzyme’s conformation was affected and hence the observed loss in enzyme activity. In the future, we will employ site‐directed mutagenesis to gain an insight whether our observed inhibition was due to the sensitivity of cysteine residues to oxidation. Support or Funding Information This work was funded by a Research Scholar Grant, RSG‐19‐075‐01‐TBE, from the American Cancer Society (C.D.S.), National Institutes of Health R00 CA187594 (C.D.S.), U54CA132384 (SDSU) & U54CA132379 (UC San Diego), MARC 5T34GM008303 (SDSU), and IMSD 5R25GM058906 (SDSU), as well as the California Metabolic Research Foundation (SDSU).

Polymerase n (PolN) is a poorly understood error‐prone polymerase that is involved in DNA repair pathways. PolN‐deficient cells are more sensitive to interstrand cross‐links (ICL) agents, suggesting that PolN is involved in repairing ICL. PolN has also been linked to cancer, including amplification in neuroendocrine prostate cancers, but its role is unknown. One of the challenges that hinders PolN studies is the poor heterologous expression of PolN. Here we seek to systematically improve the expression and purification of PolN heterologously expressed in E. coli. Plasmids with fusion partners that enhance high expression of insoluble proteins have been widely used. We employed the maltose‐binding protein (MBP) as a solubility‐enhanced protein and His tag to facilitate bacterial expression and purification. The Gateway plasmid was transformed to BL21 DE3 bacterial strain and the expression was induced by adding 0.5mM isopropyl β‐ d‐1‐thiogalactopyranoside (IPTG) after an OD600 of 0.6 was reached and kept constant at cold temperature overnight. The expression was optimized further to increase soluble protein production, including addition of arabinose and varying concentrations of both arabinose and IPTG. The induction temperature was kept at 37 degree Celsius and was kept constant for a duration of less than 10 hours. Finally, the cells were harvested and analyzed by SDS‐PAGE to assess expression levels. The higher expression level was achieved at a cold temperature in the presence of both IPTG and 1% of arabinose. To achieve higher concentration of PolN harvested, we will increase the volume of cultures that will be expressed and purification will be done using affinity chromatography.