The endoplasmic reticulum (ER) membrane of plant cells contains several enzymes responsible for the biosynthesis of a diverse range of molecules essential for plant growth and holds potential for industrial applications. Many of these enzymes are dependent on electron transfer proteins to sustain their catalytic cycles. In plants, two crucial ER-bound electron transfer proteins are cytochrome b5 and cytochrome b5 reductase, which catalyze the stepwise transfer of electrons from NADH to redox enzymes such as fatty acid desaturases, cytochrome P450s, and plant aldehyde decarbonylase. Despite the high significance of plant cytochrome b5 and cytochrome b5 reductase, they have eluded detailed characterization to date. Here, we overexpressed the full-length membrane-bound cytochrome b5 isoform B from the model plant Arabidopsis thaliana in Escherichia coli, purified the protein employing detergents as well as styrene−maleic acid (SMA) copolymers, and biochemically characterized the protein. The SMA-encapsulated cytochrome b5 exhibits a discoidal shape and the characteristic features of the active heme-bound state. We also overexpressed and purified the soluble domain of cytochrome b5 reductase from A. thaliana, establishing its activity, stability, and kinetic parameters. Further, we demonstrated that the plant cytochrome b5, purified in detergents and styrene maleic acid lipid particles (SMALPs), readily accepts electrons from the cognate plant cytochrome b5 reductase and distant electron mediators such as plant NADPH-cytochrome P450 oxidoreductase and cyanobacterial NADPH-ferredoxin reductase. We also measured the kinetic parameters of cytochrome b5 reductase for cytochrome b5. Our studies are the first to report the purification and detailed biochemical characterization of the plant cytochrome b5 and cytochrome b5 reductase from the bacterial overexpression system.

Early detection of Alzheimer’s disease (AD) is important for taking proper measures against AD pathogenesis. Acetylcholinesterase (AChE) is widely reported to be associated with the pathogenicity of AD. Here, employing the “acetylcholine-mimic” approach, we designed and synthesized a new class of naphthalimide (Naph)-based fluorogenic probes for specific detection of AChE and avoiding interference of butyrylcholinesterase (BuChE), the pseudocholinesterase. We investigated the action of the probes on Electrophorus electricus AChE, and the native human brain AChE that we expressed in Escherichia coli and purified in the active form for the first time. The probe Naph-3 exhibited a substantial fluorescence enhancement with AChE and majorly avoided BuChE. Naph-3 successfully crossed the cell membrane of the Neuro-2a cells and fluoresced upon reaction with endogenous AChE. We further established that the probe could be effectively used for screening AChE inhibitors. Our study provides a new avenue for the specific detection of AChE, which can be extended to the diagnosis of AChE-related complications.

Waning resources, massive energy consumption, ever‐deepening global warming crisis, and climate change have raised grave concerns regarding continued dependence on fossil fuels as the predominant source of energy and generated tremendous interest for developing biofuels, which are renewable. Hydrocarbon‐based ′drop‐in′ biofuels can be a proper substitute for fossil fuels such as gasoline or jet fuel. In Nature, hydrocarbons are produced by diverse organisms such as insects, plants, bacteria, and cyanobacteria. Metalloenzymes play a crucial role in hydrocarbons biosynthesis, and the past decade has witnessed discoveries of a number of metalloenzymes catalyzing hydrocarbon biosynthesis from fatty acids and their derivatives employing unprecedented mechanisms. These discoveries elucidated the enigma related to the divergent chemistries involved in the catalytic mechanisms of these metalloenzymes. There is substantial diversity in the structure, mode of action, cofactor requirement, and substrate scope among these metalloenzymes. Detailed structural analysis along with mutational studies of some of these enzymes have contributed significantly to identifying the key amino acid residues that dictate substrate specificity and catalytic intricacy. In this Review, we discuss the metalloenzymes that catalyze fatty acid‐derived hydrocarbon biosynthesis in various organisms, emphasizing the active site architecture, catalytic mechanism, cofactor requirements, and substrate specificity of these enzymes. Understanding such details is essential for successfully implementing these enzymes in emergent biofuel research through protein engineering and synthetic biology approaches.

Hydrocarbon biosynthesis has gained tremendous attention in recent years due to its implication in the development of next‐generation advanced biofuels. In nature, various organisms such as plants, insects, bacteria, and cyanobacteria produce fatty acid‐derived alka(e)nes. Most of the enzymes involved in fatty acid‐derived hydrocarbon biosynthesis are metalloenzymes, which employ divergent and intriguing catalytic mechanisms. Herein, metalloenzymes are reviewed in detail with special emphasis on their structure and mechanism. More information can be found in the Review by Debasis Das et al.

Biosynthetically produced alkenes are high-value molecules that can serve as ‘drop-in’ replacements for fossil fuels. Alkenes are also heavily used in the polymer, lubricant, and detergent industries. UndB is the only known membrane-bound fatty acid decarboxylase that catalyzes the conversion of fatty acids to terminal alkenes at the highest reported in vivo titers. However, the enzyme remains poorly understood and enigmatic. Here, we demonstrate the first-time purification of UndB and establish that it is an oxygen-dependent, non-heme diiron enzyme that engages conserved histidine residues at the active site. We also identify redox partners that support the activity of UndB and determine the enzyme's substrate specificity and kinetic properties. We detect CO2 as the co-product of the UndB-catalyzed reaction and provide the first evidence in favor of the hydrogen atom transfer (HAT) mechanism of the enzyme. Our findings decipher the biochemistry of an enigmatic metalloenzyme that catalyzes 1-alkene biosynthesis at the membrane interface with the highest known efficiency.