Dynamics-driven allosteric stimulation of diguanylate cyclase activity in a red light-regulated phytochrome.

Sensor-effector proteins integrate information from different stimuli and transform this into cellular responses. Some sensory domains, like red-light responsive bacteriophytochromes, show remarkable modularity regulating a variety of effectors. One effector domain is the GGDEF diguanylate cyclase catalyzing the formation of the bacterial second messenger cyclic-dimeric-guanosine monophosphate. While critical signal integration elements have been described for different phytochromes, a generalized understanding of signal processing and communication over large distances, roughly 100 Å in phytochrome diguanylate cyclases, is missing. Here we show that dynamics-driven allostery is key to understanding signal integration on a molecular level. We generated protein variants stabilized in their far-red-absorbing Pfr state and demonstrated by analysis of conformational dynamics using hydrogen-deuterium exchange coupled to mass spectrometry that single amino acid replacements are accompanied by altered dynamics of functional elements throughout the protein. We show that the conformational dynamics correlate with the enzymatic activity of these variants, explaining also the increased activity of a non-photochromic variant. In addition, we demonstrate the functional importance of mixed Pfr/intermediate state dimers using a fast-reverting variant that still enables wild-type-like fold-changes of enzymatic stimulation by red light. This supports the functional role of single protomer activation in phytochromes, a property that might correlate with the non-canonical mixed Pfr/intermediate-state spectra observed for many phytochrome systems. We anticipate our results to stimulate research in the direction of dynamics-driven allosteric regulation of different bacteriophytochrome-based sensor-effectors. This will eventually impact design strategies for the creation of novel sensor-effector systems for enriching the optogenetic toolbox.

Successful long-term management of spasticity in people with multiple sclerosis using a software application: Results from a randomized, controlled, multicenter study.

BACKGROUND AND PURPOSE: Successful long-term treatment of spasticity in people with multiple sclerosis (pwMS) is challenging. We investigated the effects of multidisciplinary inpatient rehabilitation (MIR) and an individualized self-training program delivered by an app on spasticity in pwMS. METHODS: First, we assessed the efficacy of 4-week MIR in ambulatory pwMS (Expanded Disability Status Scale < 7.0) with moderate to severe lower limb spasticity (defined by ≥4 points on the Numeric Rating Scale for spasticity [NRSs]) in a cohort of 115 pwMS at seven rehabilitation centers in Austria. In the case of a clinically relevant improvement in spasticity of ≥20% on the NRSs following MIR (n = 94), pwMS were randomly allocated in a 1:1 ratio to either the newly designed MS-Spasticity App or to a paper-based self-training program for 12 weeks. The primary outcome was change in NRSs (German Clinical Trials Registry DRKS00023960). RESULTS: MIR led to a significant reduction of 2.0 points on the NRSs (95% confidence interval [CI] = 2.5-2.0, p < 0.000). MIR was further associated with a statistically significant improvement in spasticity on the Modified Ashworth Scale, strength, and all mobility outcomes. Following MIR, self-training with the MS-Spasticity App was associated with a sustained positive effect on the NRSs, whereas paper-based self-training led to a worsening in spasticity (median NRSs difference = 1.0, 95% CI = 1.7-0.3, p = 0.009). The MS-Spasticity App was also associated with a significantly better adherence to self-training (95% vs. 72% completion rate, p < 0.001). CONCLUSIONS: In pwMS, MIR is able to significantly improve lower limb spasticity, strength, and mobility. Following MIR, an individually tailored antispasticity program delivered by an app leads to sustained positive long-term management.

Characterisation of sequence-structure-function space in sensor-effector integrators of phytochrome-regulated diguanylate cyclases.

Understanding the relationship between protein sequence, structure and function is one of the fundamental challenges in biochemistry. A direct correlation, however, is often not trivial since protein dynamics also play an important functional role-especially in signal transduction processes. In a subfamily of bacterial light sensors, phytochrome-activated diguanylate cyclases (PadCs), a characteristic coiled-coil linker element connects photoreceptor and output module, playing an essential role in signal integration. Combining phylogenetic analyses with biochemical characterisations, we were able to show that length and composition of this linker determine sensor-effector function and as such are under considerable evolutionary pressure. The linker length, together with the upstream PHY-specific domain, influences the dynamic range of effector activation and can even cause light-induced enzyme inhibition. We demonstrate phylogenetic clustering according to linker length, and the development of new linker lengths as well as new protein function within linker families. The biochemical characterisation of PadC homologs revealed that the functional coupling of PHY dimer interface and linker element defines signal integration and regulation of output functionality. A small subfamily of PadCs, characterised by a linker length breaking the coiled-coil pattern, shows a markedly different behaviour from other homologs. The effect of the central helical spine on PadC function highlights its essential role in signal integration as well as direct regulation of diguanylate cyclase activity. Appreciation of sensor-effector linkers as integrator elements and their coevolution with sensory modules is a further step towards the use of functionally diverse homologs as building blocks for rationally designed optogenetic tools.

The structural effect between the output module and chromophore-binding domain is a two-way street via the hairpin extension.

Signal transduction typically starts with either ligand binding or cofactor activation, eventually affecting biological activities in the cell. In red light-sensing phytochromes, isomerization of the bilin chromophore results in regulation of the activity of diverse output modules. During this process, several structural elements and chemical events influence signal propagation. In our study, we have studied the full-length bacteriophytochrome from Deinococcus radiodurans as well as a previously generated optogenetic tool where the native histidine kinase output module has been replaced with an adenylate cyclase. We show that the composition of the output module influences the stability of the hairpin extension. The hairpin, often referred as the PHY tongue, is one of the central structural elements for signal transduction. It extends from a distinct domain establishing close contacts with the chromophore binding site. If the coupling between these interactions is disrupted, the dynamic range of the enzymatic regulation is reduced. Our study highlights the complex conformational properties of the hairpin extension as a bidirectional link between the chromophore-binding site and the output module, as well as functional properties of diverse output modules.

Cord clamping beyond 3 minutes: Neonatal short-term outcomes and maternal postpartum hemorrhage.

BACKGROUND: Delaying cord clamping (CC) for 3-5 minutes reduces iron deficiency and improves neurodevelopment. Data on the effects of CC beyond 3 minutes in relation to short-term neonatal outcomes and maternal risk of postpartum hemorrhage are scarce. METHODS: This was a prospective observational study performed in two delivery departments. Pregnant women with vaginal deliveries were included. Time to CC, estimated postpartum blood loss, and perinatal data were recorded. Spearman's correlation analysis and comparisons between newborns clamped before and after 3 minutes were performed. RESULTS: In total, 904 dyads were included. The mean gestational age ± standard deviation was 40.1 ± 1.2 weeks. CC was performed at a median time of 6 minutes (range 0-23.5). Apgar scores at 5 and 10 minutes were positively correlated with time to CC (correlation coefficient .140, P < .001 and .161, < .001). There was no correlation between CC time and bilirubin level (correlation coefficient .021, P = .54). The median postpartum blood loss was 300 mL (70-2550 mL), with a negative correlation between CC time and postpartum blood loss (-0.115, P = .001). The postpartum blood loss was larger in the group clamped at ≤3 minutes (median [interquartile range] 400 mL [300-600] vs 300 mL [250-450], [P = .003]]. CONCLUSIONS: Umbilical CC times beyond 3 minutes in vaginal deliveries were not associated with negative short-term outcomes in newborns and were associated with a smaller maternal postpartum blood loss. Although CC time as long as 6 minutes could be considered as safe, further research is needed to decide the optimal timing.

Diameter Changes in Traumatic Aortic Injury: Implications for Stent-Graft Sizing.

OBJECTIVES: The aim of this study was to compare aortic diameters from admission computed tomography angiography (CTA) scans to postoperative aortic diameters in patients with traumatic aortic injury (TAI) and evaluate the influence of substantial blood loss on aortic diameter. METHODS: The aortic databases of two tertiary university centers were retrospectively screened for patients with TAI between February 2002 and February 2019. Concomitant organ injuries, bone fractures, blood loss, and clinical outcomes were evaluated. Aortic diameters were measured in CTA upon admission and were compared with the CTA before discharge at three different aortic levels (mid-ascending, 5 cm distal to the end of the stent graft, and at the celiac trunk level). RESULTS: We identified 45 patients, aged 43 (first quartile; third quartile [26; 55]) years with a TAI treated by thoracic endovascular aortic repair. The most frequent cause of TAI was a car accident (n = 24). Concomitant injuries were seen in all but one patient. Bone and pelvic fractures were seen in 40 (89%) and 15 (33%) patients, respectively. Type III aortic injury was present in 25 patients (56%). Increase of aortic diameter after stabilization was +1.7 mm (-0.6 mm; 2.5 mm; p = 0.004) at the mid-ascending aorta, +2.1 mm (0.2 mm; 3.8 mm; p < 0.001) 5 cm distal to the stent graft, and +1.5 mm (0.5 mm; 3.2 mm; p < 0.001) at the celiac trunk level. CONCLUSION: In patients with TAI, the aortic diameter is significantly reduced as compared with the aortic diameter at discharge. The reduction of aortic diameter might be caused by hemorrhagic shock and should be kept in mind for appropriate stent-graft sizing.

Distinct chromophore-protein environments enable asymmetric activation of a bacteriophytochrome-activated diguanylate cyclase.

Sensing of red and far-red light by bacteriophytochromes involves intricate interactions between their bilin chromophore and the protein environment. The light-triggered rearrangements of the cofactor configuration and eventually the protein conformation enable bacteriophytochromes to interact with various protein effector domains for biological modulation of diverse physiological functions. Excitation of the holoproteins by red or far-red light promotes the photoconversion to their far-red light-absorbing Pfr state or the red light-absorbing Pr state, respectively. Because prototypical bacteriophytochromes have a parallel dimer architecture, it is generally assumed that symmetric activation with two Pfr state protomers constitutes the signaling-active species. However, the bacteriophytochrome from Idiomarina species A28L (IsPadC) has recently been reported to enable long-range signal transduction also in asymmetric dimers containing only one Pfr protomer. By combining crystallography, hydrogen-deuterium exchange coupled to MS, and vibrational spectroscopy, we show here that Pfr of IsPadC is in equilibrium with an intermediate "Pfr-like" state that combines features of Pfr and Meta-R states observed in other bacteriophytochromes. We also show that structural rearrangements in the N-terminal segment (NTS) can stabilize this Pfr-like state and that the PHY-tongue conformation of IsPadC is partially uncoupled from the initial changes in the NTS. This uncoupling enables structural asymmetry of the overall homodimeric assembly and allows signal transduction to the covalently linked physiological diguanylate cyclase output module in which asymmetry might play a role in the enzyme-catalyzed reaction. The functional differences to other phytochrome systems identified here highlight opportunities for using additional red-light sensors in artificial sensor-effector systems.

What Is the Best Method to Achieve Safe and Precise Stent-Graft Deployment in Patients Undergoing TEVAR?

Thoracic endovascular aortic repair (TEVAR) for aortic pathologies requires sufficient landing zone of ideally more than 25 mm for safe anchoring of the stent-graft and prevention of endoleaks. In the aortic arch and at the thoracoabdominal transition, landing zone length is usually limited by the offspring of the major aortic side-branches. Exact deployment of the stent-graft to effectively use the whole length of the landing zone and to prevent occlusion of one of the side-branches is key to successful TEVAR. There are numerous techniques described to lower blood pressure and to reduce or eliminate aortic impulse to facilitate exact deployment of stent-grafts including pharmacologic blood pressure lowering, adenosine-induced asystole, inflow occlusion, and rapid pacing. Aim of this review was to assess the current literature to identify which of the techniques is best suited to prevent displacement and allow for precise placement of the stent-graft and safe balloon-molding.

Influence of the N-terminal segment and the PHY-tongue element on light-regulation in bacteriophytochromes.

Photoreceptors enable the integration of ambient light stimuli to trigger lifestyle adaptations via modulation of central metabolite levels involved in diverse regulatory processes. Red light-sensing bacteriophytochromes are attractive targets for the development of innovative optogenetic tools because of their natural modularity of coupling with diverse functionalities and the natural availability of the light-absorbing biliverdin chromophore in animal tissues. However, a rational design of such tools is complicated by the poor understanding of molecular mechanisms of light signal transduction over long distances-from the site of photon absorption to the active site of downstream enzymatic effectors. Here we show how swapping structural elements between two bacteriophytochrome homologs provides additional insight into light signal integration and effector regulation, involving a fine-tuned interplay of important structural elements of the sensor, as well as the sensor-effector linker. Facilitated by the availability of structural information of inhibited and activated full-length structures of one of the two homologs (Idiomarina species A28L phytochrome-activated diguanylyl cyclase (IsPadC)) and characteristic differences in photoresponses of the two homologs, we identify an important cross-talk between the N-terminal segment, containing the covalent attachment site of the chromophore, and the PHY-tongue region. Moreover, we highlight how these elements influence the dynamic range of photoactivation and how activation can be improved to light/dark ratios of ∼800-fold by reducing basal dark-state activities at the same time as increasing conversion in the light state. This will enable future optimization of optogenetic tools aiming at a direct allosteric regulation of enzymatic effectors.

Genotoxic potential of cellobiose.

Cellobiose is a naturally occurring only slightly sweet disaccharide that may be used as a novel food ingredient. As part of the safety evaluation of cellobiose, produced from sucrose by an enzymatic process, an Ames test and an in-vitro micronucleus test with human peripheral lymphocytes were performed according to the respective OECD Guidelines. The results demonstrate that cellobiose lacks a genotoxic potential under the conditions of these standardized assays.

Structure-guided design and functional characterization of an artificial red light-regulated guanylate/adenylate cyclase for optogenetic applications.

Genetically targeting biological systems to control cellular processes with light is the concept of optogenetics. Despite impressive developments in this field, underlying molecular mechanisms of signal transduction of the employed photoreceptor modules are frequently not sufficiently understood to rationally design new optogenetic tools. Here, we investigate the requirements for functional coupling of red light-sensing phytochromes with non-natural enzymatic effectors by creating a series of constructs featuring the Deinococcus radiodurans bacteriophytochrome linked to a Synechocystis guanylate/adenylate cyclase. Incorporating characteristic structural elements important for cyclase regulation in our designs, we identified several red light-regulated fusions with promising properties. We provide details of one light-activated construct with low dark-state activity and high dynamic range that outperforms previous optogenetic tools in vitro and expands our in vivo toolkit, as demonstrated by manipulation of Caenorhabditis elegans locomotor activity. The full-length crystal structure of this phytochrome-linked cyclase revealed molecular details of photoreceptor-effector coupling, highlighting the importance of the regulatory cyclase element. Analysis of conformational dynamics by hydrogen-deuterium exchange in different functional states enriched our understanding of phytochrome signaling and signal integration by effectors. We found that light-induced conformational changes in the phytochrome destabilize the coiled-coil sensor-effector linker, which releases the cyclase regulatory element from an inhibited conformation, increasing cyclase activity of this artificial system. Future designs of optogenetic functionalities may benefit from our work, indicating that rational considerations for the effector improve the rate of success of initial designs to obtain optogenetic tools with superior properties.

Oxidation of the FAD cofactor to the 8-formyl-derivative in human electron-transferring flavoprotein.

The heterodimeric human (h) electron-transferring flavoprotein (ETF) transfers electrons from at least 13 different flavin dehydrogenases to the mitochondrial respiratory chain through a non-covalently bound FAD cofactor. Here, we describe the discovery of an irreversible and pH-dependent oxidation of the 8α-methyl group to 8-formyl-FAD (8f-FAD), which represents a unique chemical modification of a flavin cofactor in the human flavoproteome. Furthermore, a set of hETF variants revealed that several conserved amino acid residues in the FAD-binding pocket of electron-transferring flavoproteins are required for the conversion to the formyl group. Two of the variants generated in our study, namely αR249C and αT266M, cause glutaric aciduria type II, a severe inherited disease. Both of the variants showed impaired formation of 8f-FAD shedding new light on the potential molecular cause of disease development. Interestingly, the conversion of FAD to 8f-FAD yields a very stable flavin semiquinone that exhibited slightly lower rates of electron transfer in an artificial assay system than hETF containing FAD. In contrast, the formation of 8f-FAD enhanced the affinity to human dimethylglycine dehydrogenase 5-fold, indicating that formation of 8f-FAD modulates the interaction of hETF with client enzymes in the mitochondrial matrix. Thus, we hypothesize that the FAD cofactor bound to hETF is subject to oxidation in the alkaline (pH 8) environment of the mitochondrial matrix, which may modulate electron transport between client dehydrogenases and the respiratory chain. This discovery challenges the current concepts of electron transfer processes in mitochondria.

LOVTRAP: an optogenetic system for photoinduced protein dissociation.

LOVTRAP is an optogenetic approach for reversible light-induced protein dissociation using protein A fragments that bind to the LOV domain only in the dark, with tunable kinetics and a >150-fold change in the dissociation constant (Kd). By reversibly sequestering proteins at mitochondria, we precisely modulated the proteins' access to the cell edge, demonstrating a naturally occurring 3-mHz cell-edge oscillation driven by interactions of Vav2, Rac1, and PI3K proteins.

Physical rupture of the xylem in developing sweet cherry fruit causes progressive decline in xylem sap inflow rate.

MAIN CONCLUSION: Xylem flow is progressively shut down during maturation beginning with minor veins at the stylar end and progressing to major veins and finally to bundles at the stem end. This study investigates the functionality of the xylem vascular system in developing sweet cherry fruit (Prunus avium L.). The tracers acid fuchsin and gadoteric acid were fed to the pedicel of detached fruit. The tracer distribution was studied using light microscopy and magnetic resonance imaging. The vasculature of the sweet cherry comprises five major bundles. Three of these supply the flesh; two enter the pit to supply the ovules. All vascular bundles branch into major and minor veins that interconnect via numerous anastomoses. The flow in the xylem as indexed by the tracer distribution decreases continuously during development. The decrease is first evident at the stylar (distal) end of the fruit during pit hardening and progresses basipetally towards the pedicel (proximal) end of the fruit at maturity. That growth strains are the cause of the decreased conductance is indicated by: elastic strain relaxation after tissue excision, the presence of ruptured vessels in vivo, the presence of intrafascicular cavities, and the absence of tyloses.

The family of berberine bridge enzyme-like enzymes: A treasure-trove of oxidative reactions.

Biological oxidations form the basis of life on earth by utilizing organic compounds as electron donors to drive the generation of metabolic energy carriers, such as ATP. Oxidative reactions are also important for the biosynthesis of complex compounds, i.e. natural products such as alkaloids that provide vital benefits for organisms in all kingdoms of life. The vitamin B2-derived cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) enable an astonishingly diverse array of oxidative reactions that is based on the versatility of the redox-active isoalloxazine ring. The family of FAD-linked oxidases can be divided into subgroups depending on specific sequence features in an otherwise very similar structural context. The sub-family of berberine bridge enzyme (BBE)-like enzymes has recently attracted a lot of attention due to the challenging chemistry catalyzed by its members and the unique and unusual bi-covalent attachment of the FAD cofactor. This family is the focus of the present review highlighting recent advancements into the structural and functional aspects of members from bacteria, fungi and plants. In view of the unprecedented reaction catalyzed by the family's namesake, BBE from the California poppy, recent studies have provided further insights into nature's treasure chest of oxidative reactions.

Structural and biochemical properties of LuxF from Photobacterium leiognathi.

The lux-operon of bioluminescent bacteria contains the genes coding for the enzymes required for light emission. Some species of Photobacteria feature an additional gene, luxF, which shows similarity to luxA and luxB, the genes encoding the heterodimeric luciferase. Isolated dimeric LuxF binds four molecules of an unusually derivatized flavin, i.e., 6-(3'-(R)-myristyl)-FMN (myrFMN). In the present study we have heterologously expressed LuxF in Escherichia coli BL21 in order to advance our understanding of the protein's binding properties and its role in photobacterial bioluminescence. Structure determination by X-ray crystallography confirmed that apo-LuxF possesses four preorganized binding sites, which are further optimized by adjusting the orientation of amino acid side chains. To investigate the binding properties of recombinant LuxF we have isolated myrFMN from Photobacterium leiognathi S1. We found that LuxF binds myrFMN tightly with a dissociation constant of 80±20 nM demonstrating that the purified apo-form of LuxF is fully competent in myrFMN binding. In contrast to LuxF, binding of myrFMN to luciferase is much weaker (Kd=4.0±0.4 µM) enabling LuxF to prevent inhibition of the enzyme by scavenging myrFMN. Moreover, we have used apo-LuxF to demonstrate that myrFMN occurs in all Photobacteria tested, irrespective of the presence of luxF indicating that LuxF is not required for myrFMN biosynthesis.

Muscle mass, strength and functional outcomes in critically ill patients after cardiothoracic surgery: does neuromuscular electrical stimulation help? The Catastim 2 randomized controlled trial.

BACKGROUND: The effects of neuromuscular electrical stimulation (NMES) in critically ill patients after cardiothoracic surgery are unknown. The objectives were to investigate whether NMES prevents loss of muscle layer thickness (MLT) and strength and to observe the time variation of MLT and strength from preoperative day to hospital discharge. METHODS: In this randomized controlled trial, 54 critically ill patients were randomized into four strata based on the SAPS II score. Patients were blinded to the intervention. In the intervention group, quadriceps muscles were electrically stimulated bilaterally from the first postoperative day until ICU discharge for a maximum of 14 days. In the control group, the electrodes were applied, but no electricity was delivered. The primary outcomes were MLT measured by ultrasonography and muscle strength evaluated with the Medical Research Council (MRC) scale. The secondary functional outcomes were average mobility level, FIM score, Timed Up and Go Test and SF-12 health survey. Additional variables of interest were grip strength and the relation between fluid balance and MLT. Linear mixed models were used to assess the effect of NMES on MLT, MRC score and grip strength. RESULTS: NMES had no significant effect on MLT. Patients in the NMES group regained muscle strength 4.5 times faster than patients in the control group. During the first three postoperative days, there was a positive correlation between change in MLT and cumulative fluid balance (r = 0.43, P = 0.01). At hospital discharge, all patients regained preoperative levels of muscle strength, but not of MLT. Patients did not regain their preoperative levels of average mobility (P = 0.04) and FIM score (P = 0.02) at hospital discharge, independent of group allocation. CONCLUSIONS: NMES had no effect on MLT, but was associated with a higher rate in regaining muscle strength during the ICU stay. Regression of intramuscular edema during the ICU stay interfered with measurement of changes in MLT. At hospital discharge patients had regained preoperative levels of muscle strength, but still showed residual functional disability and decreased MLT compared to pre-ICU levels in both groups. TRIAL REGISTRATION: Clinicaltrials.gov identifier NCT02391103. Registered on 7 March 2015.

Adiponectin and resistin in acute and chronic graft-vs-host disease patients undergoing allogeneic hematopoietic stem cell transplantation.

AIM: To investigate the association of adiponectin and resistin levels in patients undergoing hematopoietic stem cell transplantation (HSCT) with the clinical outcome, including the occurrence of acute and chronic graft-vs-host disease (GVHD), non-relapse mortality, and overall survival. METHODS: We prospectively collected serum samples from 40 patients undergoing either autologous (n=12; 10 male) or allogeneic (n=28; 11 male) HSCT for up to 12 months post HSCT and determined adiponectin and resistin serum concentrations using enzyme-linked immunosorbent assay. RESULTS: There were no significant differences in adiponectin levels (18.5 vs 9.3 µg/mL, P=0.071) and adiponectin/BMI ratio (0.82 vs 0.39, P=0.068) between patients with acute GVHD grades 2-4 and autologous controls. However, resistin values were significantly lower in patients with acute GVHD grades 2-4 than in autologous controls (4.6 vs 7.3 ng/mL, P=0.030). Adiponectin levels were higher in patients with chronic GVHD (n=17) than in autologous controls (13.5 vs 7.6 µg/mL, P=0.051), but the difference was not significant. Adiponectin/BMI ratio was significantly higher in patients with chronic GVHD than in autologous controls (0.59 vs 0.25, P=0.006). Patients dying from relapse also had significantly lower adiponectin levels (8.2 µg/mL) and adiponectin/BMI ratio (0.3) on admission than surviving allogeneic (15.8 µg/mL, P=0.030 and 0.7, P=0.004) and surviving autologous patients (19.2 µg/mL, P=0.031 and 0.7, P=0.021). CONCLUSION: Adiponectin and resistin levels were altered in patients with acute and chronic GVHD compared to autologous controls and were associated with overall survival and relapse mortality in patients undergoing allogeneic HSCT.

Rapid access to glycopeptide antibiotic precursor peptides coupled with cytochrome P450-mediated catalysis: towards a biomimetic synthesis of glycopeptide antibiotics.

Understanding the mechanisms underpinning glycopeptide antibiotic biosynthesis is key to the future ability to reinvent these compounds. For effective in vitro characterization of the crucial later steps of the biosynthesis, facile access to a wide range of substrate peptides as their Coenzyme A (CoA) conjugates is essential. Here we report the development of a rapid route to glycopeptide precursor CoA conjugates that affords both high yields and excellent purities. This synthesis route is applicable to the synthesis of peptide CoA-conjugates containing racemization-prone arylglycine residues: such residues are hallmarks of non-ribosomal peptide synthesis and have previously been inaccessible to peptide synthesis using Fmoc-type chemistry. We have applied this route to generate glycopeptide precursor peptides in their carrier protein-bound form as substrates to explore the specificity of the first oxygenase enzyme from vancomycin biosynthesis (OxyBvan). Our results indicate that OxyBvan is a highly promiscuous catalyst for phenolic coupling of diverse glycopeptide precursors that accepts multiple carrier protein substrates, even on carrier protein domains from alternate glycopeptide biosynthetic machineries. These results represent the first important steps in the development of an in vitro biomimetic synthesis of modified glycopeptide aglycones.