NAD+ Kinase Enzymes Are Reversible, and NAD+ Product Inhibition Is Responsible for the Observed Irreversibility of the Human Enzyme.

The NAD+ kinase (NADK) is the only known enzyme capable of phosphorylating NAD(H) to NADP(H) and therefore it plays a crucial role in maintaining NAD(P)(H) homeostasis. All domains of life contain at least one NADK gene, and the commonly investigated isoforms have been measured, or assumed, to be functionally irreversible. In 1977, the kinetics of native pigeon liver NADK were thoroughly investigated, and it was reported to exhibit reversible activity, such that ATP and NAD+ can be formed from ADP and NADP+. We hypothesized that the reverse activity of the pigeon enzyme may enable compensation of the high picolinic acid carboxylase (PC) activity present in pigeon livers, which inhibits NAD+ biosynthesis from dietary tryptophan. Here, we report the characterization of four recombinantly expressed NADKs and explore their reversible activities. Duck and cat livers have higher PC activity than pigeon livers, and the recombinant duck and cat NADKs exhibit high activity in the reverse direction. The human NADK has an affinity for NAD+ that is ∼600 times higher than the pigeon, duck, and cat isoforms, and we conclude that NAD+ serves as a potent product inhibitor for the reverse activity of the human NADK, which accounts for the observed irreversible behavior. These results demonstrate that while all four NADKs are reversible, the reverse activity of the human enzyme alone is impeded via product inhibition. This mechanism─the conversion of a reversible to a unidirectional reaction by product inhibition─may be valuable in future metabolic engineering applications.

Engineering Polyhistidine Tags on Surface Proteins of Acidithiobacillus ferrooxidans: Impact of Localization on the Binding and Recovery of Divalent Metal Cations.

Metal processing using microorganisms has many advantages including the potential for reduced environmental impacts as compared to conventional technologies.Acidithiobacillus ferrooxidansis an iron- and sulfur-oxidizing chemolithoautotroph that is known to participate in metal bioleaching, and its metabolic capabilities have been exploited for industrial-scale copper and gold biomining. In addition to bioleaching, microorganisms could also be engineered for selective metal binding, enabling new opportunities for metal bioseparation and recovery. Here, we explored the ability of polyhistidine (polyHis) tags appended to two recombinantly expressed endogenous proteins to enhance the metal binding capacity of A. ferrooxidans. The genetically engineered cells achieved enhanced cobalt and copper binding capacities, and the Langmuir isotherm captures their interaction behavior with these divalent metals. Additionally, the cellular localization of the recombinant proteins correlated with kinetic modeling of the binding interactions, where the outer membrane-associated polyHis-tagged licanantase peptide bound the metals faster than the periplasmically expressed polyHis-tagged rusticyanin protein. The selectivity of the polyHis sequences for cobalt over copper from mixed metal solutions suggests potential utility in practical applications, and further engineering could be used to create metal-selective bioleaching microorganisms.

Engineering Acinetobacter baylyi ADP1 for mevalonate production from lignin-derived aromatic compounds.

Utilization of lignin, an abundant renewable resource, is limited by its heterogenous composition and complex structure. Biological valorization of lignin provides advantages over traditional chemical processing as it occurs at ambient temperature and pressure and does not use harsh chemicals. Furthermore, the ability to biologically funnel heterogenous substrates to products eliminates the need for costly downstream processing and separation of feedstocks. However, lack of relevant metabolic networks and low tolerance to degradation products of lignin limits the application of traditional engineered model organisms. To circumvent this obstacle, we employed Acinetobacter baylyi ADP1, which natively catabolizes lignin-derived aromatic substrates through the ß-ketoadipate pathway, to produce mevalonate from lignin-derived compounds. We enabled expression of the mevalonate pathway in ADP1 and validated activity in the presence of multiple lignin-derived aromatic substrates. Furthermore, by knocking out wax ester synthesis and utilizing fed-batch cultivation, we improved mevalonate titers 7.5-fold to 1014 mg/L (6.8 mM). This work establishes a foundation and provides groundwork for future efforts to engineer improved production of mevalonate and derivatives from lignin-derived aromatics using ADP1.

Computational structure prediction provides a plausible mechanism for electron transfer by the outer membrane protein Cyc2 from Acidithiobacillus ferrooxidans.

Cyc2 is the key protein in the outer membrane of Acidithiobacillus ferrooxidans that mediates electron transfer between extracellular inorganic iron and the intracellular central metabolism. This cytochrome c is specific for iron and interacts with periplasmic proteins to complete a reversible electron transport chain. A structure of Cyc2 has not yet been characterized experimentally. Here we describe a structural model of Cyc2, and associated proteins, to highlight a plausible mechanism for the ferrous iron electron transfer chain. A comparative modeling protocol specific for trans membrane beta barrel (TMBB) proteins in acidophilic conditions (pH ~ 2) was applied to the primary sequence of Cyc2. The proposed structure has three main regimes: Extracellular loops exposed to low-pH conditions, a TMBB, and an N-terminal cytochrome-like region within the periplasmic space. The Cyc2 model was further refined by identifying likely iron and heme docking sites. This represents the first computational model of Cyc2 that accounts for the membrane microenvironment and the acidity in the extracellular matrix. This approach can be used to model other TMBBs which can be critical for chemolithotrophic microbial growth.