The methylation of unactivated carbon and phosphorus centers is a burgeoning area of biological chemistry, especially given that such reactions constitute key steps in the biosynthesis of numerous enzyme cofactors, antibiotics, and other natural products of clinical value. These kinetically challenging reactions are catalyzed exclusively by enzymes in the radical S-adenosylmethionine (SAM) superfamily and have been grouped into four classes: A, B, C, and D. Class B radical SAM (RS) methylases require a cobalamin cofactor in addition to the [4Fe-4S] cluster that is characteristic of RS enzymes. However, their poor solubility upon overexpression and their generally poor turnover has hampered detailed in vitro studies of these enzymes. It has been suggested that improper folding, possibly caused by insufficient cobalamin during their overproduction in Escherichia coli, leads to formation of inclusion bodies. Herein, we report our efforts to improve the overproduction of class B RS methylases in a soluble form by engineering a strain of E. coli to take in more cobalamin. We cloned five genes (btuC, btuE, btuD, btuF, and btuB) that encode proteins that are responsible for cobalamin uptake and transport in E. coli and coexpressed these genes with those that encode TsrM, Fom3, PhpK, and ThnK, four class B RS methylases that suffer from poor solubility during overproduction. This strategy markedly enhances cobalamin uptake into the cytoplasm and improves the solubility of the target enzymes significantly.

Introduction Critical limb ischemia (CLI) is a life and limb-threatening condition affecting 1–10% of the peripheral arterial disease (PAD) population. Traditional revascularization options are not possible for up to 50% of CLI patients, in which case, the use of cellular therapies, such as bone marrow derived mesenchymal stem cells (MSCs), hold great promise as an alternative revascularization therapy. However no randomized controlled phase 3 trials to date have demonstrated an improvement in limb salvage with cellular therapies. This may be due to either: poor cell quality (i.e. inability to generate a sufficient number of angiogenic MSCs) and/or the inadequate retention and viability of MSCs after delivery. Because concerns remain about the expansion and angiogenic potential of autologous MSCs in the CLI population, the objective of this study was to examine: the impact of our novel culture media supplement, pooled human platelet lysate (PL), in lieu of the standard fetal bovine serum (FBS), to improve the expansion potential of MSCs from CLI patients. We also characterized the in vitro angiogenic activity of MSCs from the tibia of amputated CLI limbs compared to MSCs from healthy donors. Methods MSCs from CLI patients were obtained from the tibia of patients undergoing major amputation; n=4 ischemic (ISC); n=4 ischemic and diabetic (ISC + DM). Healthy MSCs (n=4) were aspirated from the iliac crest of young and healthy donors. MSCs were successfully isolated and expanded in culture with PL or FBS. MSCs from passage 3–6 were used for phenotypic marker expression, adipogenic and osteogenic differentiation, and tested for their in vitro angiogenic activity on human microdermal endothelial cells (EC). In parallel MSCs were cultured to passage 11 for population doubling calculations. Results MSCs from both ischemic and healthy patients exhibited appropriate expression of cell surface markers and differentiation capacity. Population doublings were significantly greater for PL-stimulated compared to FBS-stimulated MSCs in all groups. The secretome of all MSCs had biologically active amounts of angiogens identified without consistent trends between groups. PL expansion did not adversely affect MSCs’ angiogenic activity compared to FBS, and both ISC and ISC + DM MSCs demonstrated similar angiogenic effects on ECs by healthy and ischemic MSCs. Conclusion PL promotes the rapid expansion of MSCs from CLI and healthy persons. Importantly, MSCs expanded from CLI patients demonstrate the desired angiogenic activity as compared to their healthy counterparts. We conclude that autologous MSCs from CLI patients can be sufficiently expanded with PL and be expected to deliver requisite angiogenic effects in vivo. We expect the improved expansion of both ISC and ISC + DM with PL to be helpful in improving the successful delivery of autologous MSCs to patients with CLI.

Fom3, a cobalamin-dependent radical S-adenosylmethionine (SAM) methylase, has recently been shown to catalyze the methylation of carbon 2″ of cytidylyl-2-hydroxyethylphosphonate (HEP-CMP) to form cytidylyl-2-hydroxypropylphosphonate (HPP-CMP) during the biosynthesis of fosfomycin, a broad-spectrum antibiotic. It has been hypothesized that a 5'-deoxyadenosyl 5'-radical (5'-dA) generated from the reductive cleavage of SAM abstracts a hydrogen atom from HEP-CMP to prime the substrate for addition of a methyl group from methylcobalamin (MeCbl); however, the mechanistic details of this reaction remain elusive. Moreover, it has been reported that Fom3 catalyzes the methylation of HEP-CMP to give a mixture of the ( S)-HPP and ( R)-HPP stereoisomers, which is rare for an enzyme-catalyzed reaction. Herein, we describe a detailed biochemical investigation of a Fom3 that is purified with 1 equiv of its cobalamin cofactor bound, which is almost exclusively in the form of MeCbl. Electron paramagnetic resonance and Mössbauer spectroscopies confirm that Fom3 contains one [4Fe-4S] cluster. Using deuterated enantiomers of HEP-CMP, we demonstrate that the 5'-dA generated by Fom3 abstracts the C2″- pro-R hydrogen of HEP-CMP and that methyl addition takes place with inversion of configuration to yield solely ( S)-HPP-CMP. Fom3 also sluggishly converts cytidylyl-ethylphosphonate to the corresponding methylated product but more readily acts on cytidylyl-2-fluoroethylphosphonate, which exhibits a lower C2″ homolytic bond-dissociation energy. Our studies suggest a mechanism in which the substrate C2″ radical, generated upon hydrogen atom abstraction by the 5'-dA, directly attacks MeCbl to transfer a methyl radical (CH) rather than a methyl cation (CH), directly forming cob(II)alamin in the process.

Cobalamin-dependent radical S-adenosylmethionine (SAM) methylases play vital roles in the de novo biosynthesis of many antibiotics, cofactors, and other important natural products, yet remain an understudied subclass of radical SAM (RS) enzymes. In addition to a [4Fe-4S] cluster that is ligated by three cysteine residues, these enzymes also contain an N-terminal cobalamin-binding domain. In vitro studies of these enzymes have been severely limited because many are insoluble or sparingly soluble upon their overproduction in E. coli. This solubility issue has led a number of groups either to purify the protein from inclusion bodies or to purify soluble protein that often lacks proper cofactor incorporation. Herein, we use TsrM as a model to describe methods that we have used to generate soluble protein that is purified in an active form with both cobalamin and [4Fe-4S] cluster cofactors bound. Additionally, we highlight the methods that we developed to characterize the enzyme following purification.

Visual impairment and intracranial pressure (VIIP) syndrome is characterized by a number of permanent ophthalmic changes, including loss of visual function. It occurs in some astronauts during long-duration spaceflight missions. Thus, understanding the pathophysiology of VIIP is currently a major priority in space medicine research. It is hypothesized that maladaptive remodeling of the optic nerve sheath (ONS), in response to microgravity-induced elevations in intracranial pressure (ICP), contributes to VIIP. However, little is known about ONS biomechanics. In this study, we developed a custom mechanical testing system that allowed for unconfined lengthening, twisting, and circumferential distension of the porcine ONS during inflation and axial loading. Data were fit to a four-fiber family constitutive equation to extract material and structural parameters. Inflation testing showed a characteristic "cross-over point" in the pressure-diameter curves under different axial loads in all samples that were tested; the cross-over pressure was [Formula: see text] mmHg ([Formula: see text]). Large sample-to-sample variations were observed in the circumferential strain, while only modest variations were observed in the circumferential stress. Multiphoton microscopy revealed that the collagen fibers of the ONS were primarily oriented axially when the tissue was loaded. The existence of this cross-over behavior is expected to be neuroprotective, as it would avoid optic nerve compression during routine changes in gaze angle, so long as ICP was within the normal range. Including these observations into computational models of VIIP will help provide insight into the pathophysiology of VIIP and could help identify risk factors and potential interventions.