Enzymes reliant on pyridoxal 5'-phosphate (PLP), the metabolically active form of vitamin B6, hold significant importance in both biology and medicine. They facilitate various biochemical reactions, particularly in amino acid and neurotransmitter metabolisms. Vitamin B6 is absorbed by organisms in its non-phosphorylated form and phosphorylated within cells by pyridoxal kinase (PLK) and pyridox-(am)-ine 5'-phosphate oxidase (PNPOx). The flavin mononucleotide dependent PNPOx enzyme converts pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate into PLP. PNPOx is vital for both biosynthesis and salvage pathways in organisms producing B6 vitamers. However, for those depending on vitamin B6 as nutrient, PNPOx participates only in the salvage pathway. Transferring PLP produced by PNPOx to client apo-enzymes is indispensable for their catalytic function, proper folding and targeting to specific organelles. PNPOx activity deficiencies due to inborn errors lead to severe neurological pathologies, particularly neonatal epileptic encephalopathy. PNPOx enzymes from different organisms maintain PLP homeostasis through highly regulated mechanisms, including structural alterations throughout the catalytic cycle and allosteric PLP binding influencing substrate transformation at the active site. Despite shared features, species-specific regulations are evident among PNPOx enzymes.

Asymmetric reduction of enoates, imines and ketones are among the most important reactions in biocatalysis. These reactions are routinely conducted using enzymes that use nicotinamide cofactors as reductants. The deazaflavin cofactor F₄₂₀ also has electrochemical properties that make it suitable as an alternative to nicotinamide cofactors for use in asymmetric reduction reactions. However, cofactor F₄₂₀-dependent enzymes remain under-explored as a resource for biocatalysis. In this review, we consider the cofactor F₄₂₀-dependent enzyme families with greatest potential for the discovery of new biocatalysts: the flavin/deazaflavin-dependent oxidoreductases (FDORs) and the luciferase-like hydride transferases (LLHTs). We discuss characterized F₄₂₀-dependent reductions that have potential for adaptation for biocatalysis, and we consider the enzymes best suited for use in the reduction of oxidized cofactor F₄₂₀ to allow cofactor recycling in situ. We also discuss recent advances in the production of cofactor F₄₂₀ and its functional analog F_O-5’- phosphate, which remains an impediment to the adoption of this family of enzymes for industrial biocatalytic processes. Finally, we discuss the prospects for the use of this cofactor and dependent enzymes as a resource for industrial biocatalysis.

Growing cells is the typical mode of operation in many aspects of biotechnology and metabolic engineering. This comes about due to cell growth processes creating a driving force that pull metabolic flux along different metabolic pathways, that indirectly help move substrate to product. But, there is an alternative mode of operation that uses resting (non-growing) cells to achieve similar or even higher productivities. In general, resting cells are provided with carbon substrates for biocatalytic reactions but starved of nitrogen or phosphorus. Such resting cells have been usefully employed in many forms of biocatalysis and biotransformation, with or without cofactor regeneration. However, much remains unknown about the transcriptome and metabolome of resting cells in biotransformation settings. This short writeup provides the backdrop of resting cells in biocatalysis, documents their use in biotransformation with application examples, and identifies research gaps that could be filled with contemporary RNA-seq and mass spectrometry proteomics technology. Overall, utility of resting cells in biocatalysis and the extant knowledge gap in their fundamental physiology are highlighted in this resource.

Although cofactor regeneration is an established system in biocatalysis, work remains in developing new and alternative cofactor regeneration systems with greater efficiency, ease of use, and higher atom economy. In addition, cofactor regeneration system only works if the cofactor regeneration reaction operates at similar kinetics compared to the biotransformation reaction. This meant that only specific cofactor regeneration system is capable of coupling with particular biotransformation reaction. This then leaves open the field for the development of a plethora of alternative cofactor regeneration systems each capable of coupling with different biotransformation reaction of different kinetics. This short write-up examines the possibility of tapping on the NADH regenerated from a two-step ethylene glycol utilization pathway. Current knowledge suggests that this angle has not been explored; thereby, opening up possibilities for future experimental investigations into the feasibility of coupling ethylene glycol utilization pathway with biotransformation reaction as a coupled cofactor regeneration system.

This review article deals with the pathways of cellular and global molybdate distribution in plants, especially with a full overview for the model plant Arabidopsis thaliana. In its oxidized state as bioavailable molybdate, molybdenum can be absorbed from the environment. Especially in higher plants, molybdenum is indispensable as part of the molybdenum cofactor (Moco), which is responsible for functionality as a prosthetic group in a variety of essential enzymes like nitrate reductase and sulfite oxidase. Therefore, plants need mechanisms for molybdate import and transport within the organism, which is accomplished via high-affinity molybdate transporter (MOT) localized in different cells and membranes. Two different MOT-families were identified. Especially legumes like Glycine max or Medicago truncatula have an increased numbers of MOT1-family members for supplying their symbionts with molybdate for nitrogenase activity. In Arabidopsis thaliana especially, the complete way of molybdate through the plant is traceable. Not only the uptake from soil by MOT1.1 and its distribution to leaves, flowers and seeds by MOT2-family members was identified, but also inside the cell the transport trough the cytoplasm and the vacuolar storage mechanisms depending on glutathione were described. Finally, supplying the Moco biosynthesis complex by MOT1.2 and MOT2.1 was demonstrated.