Acetyl Phosphate Research Articles

Impacts of Pta-AckA pathway on CPS biosynthesis and type 3 fimbriae expression in Klebsiella pneumoniae

BackgroundKlebsiella pneumoniae is a Gram-negative bacterium that can cause infections, especially in individuals with diabetes. Recently, more hypervirulent strains have emerged, posing a threat even to healthy individuals. Understanding how K. pneumoniae regulates its virulence factors is crucial. Acetyl-phosphate (AcP) is essential for bacterial metabolism and can affect virulence factor expression. However, the role of the Pta-AckA pathway, which regulates AcP levels, in K. pneumoniae pathogenesis remains unclear. MethodsDeletion mutants lacking the pta and ackA, involved in AcP production and hydrolysis, were generated in K. pneumoniae CG43S3. Their effects on AcP levels, the patterns of global acetylated protein, capsular polysaccharide (CPS) amount, serum resistance, type 3 fimbriae expression, biofilm formation, and virulence in G. mellonella larva were assessed. ResultsDeletion of ackA in K. pneumoniae CG43S3 led to AcP accumulation, while pta deletion abolished AcP synthesis when grown in TB7+1 % glucose. This pathway influenced global protein acetylation, with pta deletion decreasing acetylation and ackA deletion increasing it. Additionally, pta deletion decreased the CPS amount, serum resistance, and type 3 fimbriae expression, while ackA deletion increased these factors. Furthermore, deleting pta and ackA attenuated the infected larva's virulence and death rate. ConclusionOur findings highlight the critical role of the Pta-AckA pathway in K. pneumoniae pathogenesis. This pathway regulates AcP levels, global protein acetylation, CPS production, serum resistance, and type 3 fimbriae expression, ultimately impacting virulence. The information provides insights into potential therapeutic targets for combating K. pneumoniae infection.

The associations between dysregulation of human blood metabolites and lung cancer risk: evidence from genetic data

BackgroundMetabolic dysregulation is recognized as a significant hallmark of cancer progression. Although numerous studies have linked specific metabolic pathways to cancer incidence, the causal relationship between blood metabolites and lung cancer risk remains unclear.MethodsGenomic data from 29,266 lung cancer patients and 56,450 control individuals from the Transdisciplinary Research in Cancer of the Lung and the International Lung Cancer Consortium (TRICL-ILCCO) were utilized, and findings were replicated using additional data from the FinnGen consortium. The analysis focused on the associations between 486 blood metabolites and the susceptibility to overall lung cancer and its three major clinical subtypes. Various Mendelian randomization methods, including inverse-variance weighting, weighted median estimation, and MR-Egger regression, were employed to ensure the robustness of our findings.ResultsA total of 19 blood metabolites were identified with significant associations with lung cancer risk. Specifically, oleate (OR per SD = 2.56, 95% CI: 1.51 to 4.36), 1-arachidonoylglyceropholine (OR = 1.79, 95% CI: 1.22 to 2.65), and arachidonate (OR = 1.67, 95% CI: 1.16 to 2.40) were associated with a higher risk of lung cancer. Conversely, 1-linoleoylglycerophosphoethanolamine (OR = 0.57, 95% CI: 0.40 to 0.82), ADpSGEGDFXAEGGGVR, a fibrinogen cleavage peptide (OR = 0.60, 95% CI: 0.47 to 0.77), and isovalerylcarnitine (OR = 0.62, 95% CI: 0.49 to 0.78) were associated with a lower risk of lung cancer. Notably, isoleucine (OR = 9.64, 95% CI: 2.55 to 36.38) was associated with a significantly higher risk of lung squamous cell cancer, while acetyl phosphate (OR = 0.11, 95% CI: 0.01 to 0.89) was associated with a significantly lower risk of small cell lung cancer.ConclusionThis study reveals the complex relationships between specific blood metabolites and lung cancer risk, highlighting their potential as biomarkers for lung cancer prevention, screening, and treatment. The findings not only deepen our understanding of the metabolic mechanisms of lung cancer but also provide new insights for future treatment strategies.

Acetyl-CoA synthetase activity is enzymatically regulated by lysine acetylation using acetyl-CoA or acetyl-phosphate as donor molecule

The AMP-forming acetyl-CoA synthetase is regulated by lysine acetylation both in bacteria and eukaryotes. However, the underlying mechanism is poorly understood. The Bacillus subtilis acetyltransferase AcuA and the AMP-forming acetyl-CoA synthetase AcsA form an AcuA•AcsA complex, dissociating upon lysine acetylation of AcsA by AcuA. Crystal structures of AcsA from Chloroflexota bacterium in the apo form and in complex with acetyl-adenosine-5′-monophosphate (acetyl-AMP) support the flexible C-terminal domain adopting different conformations. AlphaFold2 predictions suggest binding of AcuA stabilizes AcsA in an undescribed conformation. We show the AcuA•AcsA complex dissociates upon acetyl-coenzyme A (acetyl-CoA) dependent acetylation of AcsA by AcuA. We discover an intrinsic phosphotransacetylase activity enabling AcuA•AcsA generating acetyl-CoA from acetyl-phosphate (AcP) and coenzyme A (CoA) used by AcuA to acetylate and inactivate AcsA. Here, we provide mechanistic insights into the regulation of AMP-forming acetyl-CoA synthetases by lysine acetylation and discover an intrinsic phosphotransacetylase allowing modulation of its activity based on AcP and CoA levels.

A network of acetyl phosphate-dependent modification modulates c-di-AMP homeostasis in Actinobacteria.

Cyclic purine nucleotides are important signal transduction molecules across all domains of life. 3',5'-cyclic di-adenosine monophosphate (c-di-AMP) has roles in both prokaryotes and eukaryotes, while the signals that adjust intracellular c-di-AMP and the molecular machinery enabling a network-wide homeostatic response remain largely unknown. Here, we present evidence for an acetyl phosphate (AcP)-governed network responsible for c-di-AMP homeostasis through two distinct substrates, the diadenylate cyclase DNA integrity scanning protein (DisA) and its newly identified transcriptional repressor, DasR. Correspondingly, we found that AcP-induced acetylation exerts these regulatory actions by disrupting protein multimerization, thus impairing c-di-AMP synthesis via K66 acetylation of DisA. Conversely, the transcriptional inhibition of disA was relieved during DasR acetylation at K78. These findings establish a pivotal physiological role for AcP as a mediator to balance c-di-AMP homeostasis. Further studies revealed that acetylated DisA and DasR undergo conformational changes that play crucial roles in differentiation. Considering the broad distribution of AcP-induced acetylation in response to environmental stress, as well as the high conservation of the identified key sites, we propose that this unique regulation of c-di-AMP homeostasis may constitute a fundamental property of central circuits in Actinobacteria and thus the global control of cellular physiology.IMPORTANCESince the identification of c-di-AMP is required for bacterial growth and cellular physiology, a major challenge is the cell signals and stimuli that feed into the decision-making process of c-di-AMP concentration and how that information is integrated into the regulatory pathways. Using the bacterium Saccharopolyspora erythraea as a model, we established that AcP-dependent acetylation of the diadenylate cyclase DisA and its newly identified transcriptional repressor DasR is involved in coordinating environmental and intracellular signals, which are crucial for c-di-AMP homeostasis. Specifically, DisA acetylated at K66 directly inactivates its diadenylate cyclase activity, hence the production of c-di-AMP, whereas DasR acetylation at K78 leads to increased disA expression and c-di-AMP levels. Thus, AcP represents an essential molecular switch in c-di-AMP maintenance, responding to environmental changes and possibly hampering efficient development. Therefore, AcP-mediated posttranslational processes constitute a network beyond the usual and well-characterized synthetase/hydrolase governing c-di-AMP homeostasis.

Bacterial protein acetylation: mechanisms, functions, and methods for study.

Lysine acetylation is an evolutionarily conserved protein modification that changes protein functions and plays an essential role in many cellular processes, such as central metabolism, transcriptional regulation, chemotaxis, and pathogen virulence. It can alter DNA binding, enzymatic activity, protein-protein interactions, protein stability, or protein localization. In prokaryotes, lysine acetylation occurs non-enzymatically and by the action of lysine acetyltransferases (KAT). In enzymatic acetylation, KAT transfers the acetyl group from acetyl-CoA (AcCoA) to the lysine side chain. In contrast, acetyl phosphate (AcP) is the acetyl donor of chemical acetylation. Regardless of the acetylation type, the removal of acetyl groups from acetyl lysines occurs only enzymatically by lysine deacetylases (KDAC). KATs are grouped into three main superfamilies based on their catalytic domain sequences and biochemical characteristics of catalysis. Specifically, members of the GNAT are found in eukaryotes and prokaryotes and have a core structural domain architecture. These enzymes can acetylate small molecules, metabolites, peptides, and proteins. This review presents current knowledge of acetylation mechanisms and functional implications in bacterial metabolism, pathogenicity, stress response, translation, and the emerging topic of protein acetylation in the gut microbiome. Additionally, the methods used to elucidate the biological significance of acetylation in bacteria, such as relative quantification and stoichiometry quantification, and the genetic code expansion tool (CGE), are reviewed.

Quantitative Analysis of the Human Semen Phosphorometabolome by 31P-NMR.

Phosphorus-containing metabolites occupy a prominent position in cell pathways. The phosphorometabolomic approach in human sperm samples will deliver valuable information as new male fertility biomarkers could emerge. This study analyzed, by 31P-NMR, seminal plasma and whole semen from asthenozoospermic and normozoospermic samples (71% vs. 27% and 45% vs. 17%, total and progressive sperm motility, respectively), and also ejaculates from healthy donors. At least 16 phosphorus-containing metabolites involved in central energy metabolism and phospholipid, nucleotide, and nicotinamide metabolic pathways were assigned and different abundances between the samples with distinct sperm quality was detected. Specifically, higher levels of phosphocholine, glucose-1-phosphate, and to a lesser degree, acetyl phosphate were found in the asthenozoospermic seminal plasma. Notably, the phosphorometabolites implicated in lipid metabolism were highlighted in the seminal plasma, while those associated with carbohydrate metabolism were more abundant in the spermatozoa. Higher levels of phosphocholine, glucose-1-phosphate, and acetyl phosphate in the seminal plasma with poor quality suggest their crucial role in supporting sperm motility through energy metabolic pathways. In the seminal plasma, phosphorometabolites related to lipid metabolism were prominent; however, spermatozoa metabolism is more dependent on carbohydrate-related energy pathways. Understanding the presence and function of sperm phosphorylated metabolites will enhance our knowledge of the metabolic profile of healthy human sperm, improving assessment and differential diagnosis.

Mild hydrolysis of chemically stable valerolactams by a biocatalytic ATP-dependent system fueled by metaphosphate.

Medium-sized 5- and 6-membered ring lactams are molecules with remarkable stability, in contrast to smaller β-lactams. As monomers, they grant access to nylon-4 and nylon-5, which are alternative polyamides to widespread caprolactam-based nylon-6. Chemical hydrolysis of monocyclic γ- and δ-lactams to the corresponding amino acids requires harsh reaction conditions and up to now, no mild (enzymatic) protocol has been reported. Herein, the biocatalytic potential of a pair of heterologously expressed bacterial ATP-dependent oxoprolinases - OplA and OplB - was exploited. Strong activity in the presence of excess of ATP was monitored on δ-valerolactam and derivatives thereof, while trace activity was detected on γ-butyrolactam. An ATP recycling system based on cheap Graham's salt (sodium metaphosphate) and a polyphosphate kinase allowed the use of catalytic amounts of ATP, leading to up to full conversion of 10 mM δ-valerolactam at 30 °C in aqueous medium. Further improvements were obtained by co-expressing OplA and OplB using the pETDuet1 vector, a strategy which enhanced the soluble expression yield and the protein stability. Finally, a range of phosphodonors was investigated in place of ATP. With acetyl phosphate and carbamoyl phosphate, turnover numbers up to 176 were reached, providing hints on a possible mechanism, which was studied by 31P-NMR.

Influence of Microbiota-Related Metabolites Associated with Inflammation and Sepsis on the Peroxidase Activity of Cyclooxygenase in Healthy Human Monocytes and Acute Monocytic Leukemia Cells.

The human microbiota produces metabolites that can enter the bloodstream and exert systemic effects on various functions in both healthy and pathological states. We have studied the participation of microbiota-related metabolites in bacterial infection by examining their influence on the activity of cyclooxygenase (COX) as a key enzyme of inflammation. The influence of aromatic microbial metabolites, derivatives of phenylalanine (phenylpropionic acid, PPA), tyrosine (4-hydroxyphenyllactic acid, HPLA), and tryptophan (indolacetic acids, IAA), the concentrations of which in the blood change notably during sepsis, was evaluated. Also, the effect of itaconic acid (ITA) was studied, which is formed in macrophages under the action of bacterial lipopolysaccharides (LPS) and appears in the blood in the early stages of infection. Metabiotic acetyl phosphate (AcP) as a strong acetylating agent was also tested. The activity of COX was measured via the TMPD oxidation colorimetric assay using the commercial pure enzyme, cultured healthy monocytes, and the human acute monocytic leukemia cell line THP-1. All metabolites in the concentration range of 100-500 μM lowered the activity of COX. The most pronounced inhibition was observed on the commercial pure enzyme, reaching up to 40% in the presence of AcP and 20-30% in the presence of the other metabolites. On cell lysates, the effect of metabolites was preserved, although it significantly decreased, probably due to their interaction with other targets subject to redox-dependent and acetylation processes. The possible contribution of the redox-dependent action of microbial metabolites was confirmed by assessing the activity of the enzyme in the presence of thiol reagents and in model conditions, when the COX-formed peroxy intermediate was replaced with tert-butyl hydroperoxide (TBH). The data show the involvement of the microbial metabolites in the regulation of COX activity, probably due to their influence on the peroxidase activity of the enzyme.

Elevated acetate kinase (ackA) gene expression, activity, and biofilm formation observed in methicillin-resistant strains of Staphylococcus aureus (MRSA)

BackgroundStaphylococcus aureus spreads its infections through biofilms. This usually happens in the stationary phase of S. aureus growth where it utilizes accumulated acetate as a carbon source via the phosphotrans-acetylase-acetate kinase (Pta-Ack) pathway. In which acetate kinase (ackA) catalyzes the substrate-level phosphorylation, a vital secondary energy-yielding pathway that promotes biofilms formation aids bacterium survival in hostile environments. In this study, we describe the cloning, sequencing, and expression of S. aureus ackA gene. The expression analysis of ackA gene in methicillin-resistant strains of S. aureus (MRSA) correlates with ackA activity and biofilm units. The uniqueness of ackA was analyzed by using in silico methods. ResultsElevated ackA gene expression was observed in MRSA strains, which correlates with increased ackA activity and biofilm units, explaining ackA role in MRSA growth and pathogenicity. The pure recombinant acetate kinase showed a molecular weight of 44 kDa, with enzyme activity of 3.35 ± 0.05 μM/ml/min. The presence of ACKA-1, ACKA-2 sites, one ATP, and five serine/threonine-protein kinase sites in the ackA gene (KC954623.1) indicated that acetyl phosphate production is strongly controlled. The comparative structural analysis of S. aureus ackA with ackA structures of Mycobacterium avium (3P4I) and Salmonella typhimurium (3SLC) exhibited variations as indicated by the RMSD values 1.877 Å and 2.141 Å respectively, explaining why ackA functions are differently placed in bacteria, concurring its involvement in S. aureus pathogenesis. ConclusionsOverall findings of this study highlight the correlation of ackA expression profoundly increases survival capacity through biofilm formation, which is a pathogenic factor in MRSA and plays a pivotal role in infection spreading.

Metal/ADP Complexes Promote Phosphorylation of Ribonucleotides.

Under enzyme catalysis, adenosine triphosphate (ATP) transfers a phosphoryl group to canonical ribonucleotide diphosphates (NDPs) to form ribonucleotide triphosphates (NTPs), the direct biosynthetic precursors to RNA. However, it remains unclear whether the phosphorylation of NDPs could have occurred in water before enzymes existed and why an adenosine derivative, rather than another canonical NTP, typically performs this function. Here, we show that adenosine diphosphate (ADP) in the presence of Fe3+ or Al3+ promotes phosphoryl transfer from acetyl phosphate to all canonical NDPs to produce their corresponding NTP in water at room temperature and in the absence of enzymes. No other NDPs were found to promote phosphorylation, giving insight into why adenosine derivatives specifically became used for this purpose in biology. The metal-ADP complexes also promote phosphoryl transfer to ribonucleoside monophosphates (NMPs) to form a mixture of the corresponding NDPs and NTPs, albeit less efficiently. This work represents a rare example in which a single nucleotide carries out a function critical to biology without enzymes. ADP-metal complexes may have played an important role in nucleotide phosphorylation in prebiotic chemistry.

Global profiling of ribosomal protein acetylation reveals essentiality of acetylation homeostasis in maintaining ribosome assembly and function.

Acetylation is a global post-translational modification that regulates various cellular processes. Bacterial acetylomic studies have revealed extensive acetylation of ribosomal proteins. However, the role of acetylation in regulating ribosome function remains poorly understood. In this study, we systematically profiled ribosomal protein acetylation and identified a total of 289 acetylated lysine residues in 52 ribosomal proteins (r-proteins) from Salmonella Typhimurium. The majority of acetylated lysine residues of r-proteins were found to be regulated by both acetyltransferase Pat and metabolic intermediate acetyl phosphate. Our results show that acetylation plays a critical role in the assembly of the mature 70S ribosome complex by modulating r-proteins binding to rRNA. Moreover, appropriate acetylation is important for the interactions between elongation factors and polysomes, as well as regulating ribosome translation efficiency and fidelity. Dysregulation of acetylation could alter bacterial sensitivity to ribosome-targeting antibiotics. Collectively, our data suggest that the acetylation homeostasis of ribosomes is crucial for their assembly and function. Furthermore, this mechanism may represent a universal response to environmental signals across different cell types.

Acetylation of K188 and K192 inhibits the DNA-binding ability of NarL to regulate Salmonella virulence.

Salmonella infection significantly increases nitrate levels in the intestine, immune cells, and immune organs of the host, and it can exploit nitrate as an electron acceptor to enhance its growth. In the presence of nitrate or nitrite, NarL, a regulatory protein of the Nar two-component system, is activated and regulates a number of genes involved in nitrate metabolism. However, research on NarL at the post-translational level is limited. In this study, we demonstrate that the DNA-binding sites K188 and 192 of NarL can be acetylated by bacterial metabolite acetyl phosphate and that the degree of acetylation has a considerable influence on the regulatory function of NarL. Specifically, acetylation of NarL negatively regulates the transcription of narG, narK, and napF, which affects the utilization of nitrate in Salmonella. Besides, both cell and mouse models show that acetylated K188 and K192 result in attenuated replication in RAW 264.7 cells, as well as impaired virulence in mouse model. Together, this research identifies a novel NarL acetylation mechanism that regulates Salmonella virulence, providing a new insight and target for salmonellosis treatment.IMPORTANCESalmonella is an important intracellular pathogen that can cause limited gastroenteritis and self-limiting gastroenteritis in immunocompetent humans. Nitrate, the highest oxidation state form of nitrogen, is critical in the formation of systemic infection in Salmonella. It functions as a signaling molecule that influences Salmonella chemotaxis, in addition to acting as a reduced external electron acceptor for Salmonella anaerobic respiration. NarL is an essential regulatory protein involved in nitrate metabolism in Salmonella, and comprehending its regulatory mechanism is necessary. Previous research has linked NarL phosphorylation to the formation of its dimer, which is required for NarL to perform its regulatory functions. Our research demonstrated that acetylation also affects the regulatory function of NarL. We found that acetylation affects Salmonella pathogenicity by weakening the ability of NarL to bind to the target sequence, further refining the mechanism of the anaerobic nitrate respiration pathway.

Using a synthetic machinery to improve carbon yield with acetylphosphate as the core

In microbial cell factory, CO2 release during acetyl-CoA production from pyruvate significantly decreases the carbon atom economy. Here, we construct and optimize a synthetic carbon conserving pathway named as Sedoheptulose-1,7-bisphosphatase Cycle with Trifunctional PhosphoKetolase (SCTPK) in Escherichia coli. This cycle relies on a generalist phosphoketolase Xfspk and converts glucose into the stoichiometric amounts of acetylphosphate (AcP). Furthermore, genetic circuits responding to AcP positively or negatively are created. Together with SCTPK, they constitute a gene-metabolic oscillator that regulates Xfspk and enzymes converting AcP into valuable chemicals in response to intracellular AcP level autonomously, allocating metabolic flux rationally and improving the carbon atom economy of bioconversion process. Using this synthetic machinery, mevalonate is produced with a yield higher than its native theoretical yield, and the highest titer and yield of 3-hydroxypropionate via malonyl-CoA pathway are achieved. This study provides a strategy for improving the carbon yield of microbial cell factories.

Multilayer precision-based screening of potential inhibitors targeting Mycobacterium tuberculosis acetate kinase using in silico approaches

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), remains a major threat to global health, with the emergence of multi-drug and extensively drug-resistant strains posing a serious challenge. Thereby, understanding the molecular basis of MTB virulence and disease pathogenesis is critical for developing effective therapeutic strategies. Targeting proteins involved in central metabolism has been recognized as a promising therapeutic approach to combat MTB. In this regard, the enzyme AckA of the acetate metabolic pathway which produces acetate from acetyl phosphate, is an important drug target for various pathogenic organisms. Therefore, this study aimed to identify potential AckA inhibitors through in silico methods, including molecular modeling, molecular dynamics simulation (MDS), and high-throughput virtual screening (HTVS) followed by ADMETox, MMGBSA, Density Functional Theory (DFT) calculations. HTVS of one million compounds from the ZINC database against AckA resulted in the top five hits (ZINC82048449, ZINC1219737510, ZINC1771921358, ZINC119699567, and ZINC1427100376) with better binding affinity and optimal binding free energy. MDS studies on complexes revealed that key residues, Asn195, Asp266, Phe267, Gly314, and Asn318 played a significant role in stable interactions of the top-ranked compounds to AckA. These outcomes provide insights into the optimal binding of the leads to inhibit the acetate pathway and aid in the rational design of novel therapeutic agents. Thus, the identified leads may act as promising compounds for targeting AckA and may serve as a potential therapeutic modality for treating TB. Our findings offer valuable insights into the inhibition of the acetate pathway, while also serving as a blueprint for rational drug design. The identified leads hold promise as compelling compounds for targeting AckA, thereby offering a potential therapeutic avenue for tackling TB. Thus, our study uncovers a pathway toward promising TB therapeutics by elucidating AckA inhibitors. By leveraging in silico methodologies, potent compounds that hold the potential to thwart AckA's role in MTB's acetate pathway have been unveiled. This breakthrough fosters optimism in the quest for novel and effective TB treatments, addressing a global health challenge with renewed vigor.

On the potential roles of phosphorus in the early evolution of energy metabolism.

Energy metabolism in extant life is centered around phosphate and the energy-dense phosphoanhydride bonds of adenosine triphosphate (ATP), a deeply conserved and ancient bioenergetic system. Yet, ATP synthesis relies on numerous complex enzymes and has an autocatalytic requirement for ATP itself. This implies the existence of evolutionarily simpler bioenergetic pathways and potentially primordial alternatives to ATP. The centrality of phosphate in modern bioenergetics, coupled with the energetic properties of phosphorylated compounds, may suggest that primordial precursors to ATP also utilized phosphate in compounds such as pyrophosphate, acetyl phosphate and polyphosphate. However, bioavailable phosphate may have been notably scarce on the early Earth, raising doubts about the roles that phosphorylated molecules might have played in the early evolution of life. A largely overlooked phosphorus redox cycle on the ancient Earth might have provided phosphorus and energy, with reduced phosphorus compounds potentially playing a key role in the early evolution of energy metabolism. Here, we speculate on the biological phosphorus compounds that may have acted as primordial energy currencies, sources of environmental energy, or sources of phosphorus for the synthesis of phosphorylated energy currencies. This review encompasses discussions on the evolutionary history of modern bioenergetics, and specifically those pathways with primordial relevance, and the geochemistry of bioavailable phosphorus on the ancient Earth. We highlight the importance of phosphorus, not only in the form of phosphate, to early biology and suggest future directions of study that may improve our understanding of the early evolution of bioenergetics.

Reactive architecture profiling with a methyl acyl phosphate electrophile

Activity-based protein profiling has facilitated the study of the activity of enzymes in proteomes, inhibitor development, and identification of enzymes that share mechanistic and active-site architectural features. Since methyl acyl phosphate monoesters act as electrostatically selective anionic electrophiles for the covalent modification of nucleophiles that reside adjacent to cationic sites in proteins, we synthesized methyl hex-5-ynoyl phosphate (MHP) to broadly target such protein architectures. After treating the soluble proteome of Paucimonas lemoignei with MHP, biotinylating the resulting acylated proteins using click chemistry, enriching the protein adducts using streptavidin, and analyzing the proteins by LC-MS/MS, a set of 240 enzymes and 132 non-enzyme proteins were identified for a wide spectrum of biological processes and from all 7 enzyme classes. Among those enzymes identified, β-hydroxybutyrate dehydrogenase (PlHBDH) and CTP synthase (E. coli orthologue, EcCTPS) were purified as recombinant enzymes and their rates of inactivation and sites of modification by MHP and methyl acetyl phosphate (MAP) were characterized. MHP reacted more slowly with these proteins than MAP but exhibited greater specificity, despite its lack of multiple binding determinants. Generally, MAP modified more surface residues than MHP. MHP specifically modified Ser 146, Lys 156, and Lys 163 at the active site of PlHBDH. MHP and MAP modified numerous residues of EcCTPS with CTP furnishing the greatest level of protection against MHP- and MAP-dependent modification and inactivation, respectively, followed by ATP and glutamine. Overall, MHP served as an effective probe to identify proteins that are potentially amenable to inhibition by methyl acyl phosphates.

Acetyl Phosphate Acetylates Proteins of Streptomyces coelicolor M-145

Acetyl Phosphate Acetylates Proteins of Streptomyces coelicolor M-145

A meet-up of acetyl phosphate and c-di-GMP modulates BldD activity for development and antibiotic production.

Actinobacteria are ubiquitous bacteria undergoing complex developmental transitions coinciding with antibiotic production in response to stress or nutrient starvation. This transition is mainly controlled by the interaction between the second messenger c-di-GMP and the master repressor BldD. To date, the upstream factors and the global signal networks that regulate these intriguing cell biological processes remain unknown. In Saccharopolyspora erythraea, we found that acetyl phosphate (AcP) accumulation resulting from environmental nitrogen stress participated in the regulation of BldD activity through cooperation with c-di-GMP. AcP-induced acetylation of BldD at K11 caused the BldD dimer to fall apart and dissociate from the target DNA and disrupted the signal transduction of c-di-GMP, thus governing both developmental transition and antibiotic production. Additionally, practical mutation of BldDK11R bypassing acetylation regulation could enhance the positive effect of BldD on antibiotic production. The study of AcP-dependent acetylation is usually confined to the control of enzyme activity. Our finding represents an entirely different role of the covalent modification caused by AcP, which integrated with c-di-GMP signal in modulating the activity of BldD for development and antibiotic production, coping with environmental stress. This coherent regulatory network might be widespread across actinobacteria, thus has broad implications.

Biocatalytic Production of Bioactive Dipeptides

Dipeptides are a promising substance class with a large variety of bioactivities. N‐terminal ar‐ ginyl dipeptides (Arg‐X) have been shown to exhibit both salt‐taste enhancing (Arg‐Ser among others) and antihypertensive properties (Arg‐Phe). A novel biocatalytic strategy for the specific synthesis of these dipeptides is the application of L‐amino acid ligases like RizA from B. subtilis, which enables production of Arg‐X dipeptides directly from their amino acids. However, challenges for an industrial application are the dependence on the expensive cofactor ATP (adenosine triphosphate), low product yields, formation of the undesired side product Arg Arg and the potentially high enzyme costs.In order to reduce the need for ATP, a strategy for ATP regeneration from acetyl phosphate was established using acetate kinase from E. coli (AckA). Up to 5.9 g/L Arg‐Ser were produced, corresponding to an ATP efficiency of 23 g Arg‐Ser per gram ATP (without regeneration max. 0.5 g Arg‐Ser/g ATP). Next, variants of RizA were created to alter the substrate specificity. A total of 21 RizA variants were created by site‐directed mutagenesis of eight positions in the substrate binding pocket. Additional 14 variants were created by combination of different mutations. Variants with improved activity and specificity were identified for all five examined amino acid combinations (Arg + Arg, Asp, Ser, Ala, Phe). The largest change was observed for K83F_S156A, which improved the product formation of Arg‐Phe six‐fold and improved specificity more than tenfold. Finally, co immobilization of RizA and selected variants with AckA was performed to enable reusability of this biocatalytic system. Immobilisates of T81F_A158S produced up to 3.0 g/L Arg Ser and immobilisates of K83F_S156A produced up to 3.8 g/L Arg‐Phe and could both be reused several times.This thesis represents the first extensive scientific investigation into establishing ATP regeneration for an L‐amino acid ligase and co‐immobilization of such a biocatalytic system, and substantially expanded the knowledge about engineering the substrate specificity of L amino acid ligases.

Identification of Serum Metabolites as Prognostic Biomarkers Following Spinal Cord Injury: A Pilot Study

The assessment, management, and prognostication of spinal cord injury (SCI) mainly rely upon observer-based ordinal scales measures. 1H nuclear magnetic resonance (NMR) spectroscopy provides an effective approach for the discovery of objective biomarkers from biofluids. These biomarkers have the potential to aid in understanding recovery following SCI. This proof-of-principle study determined: (a) If temporal changes in blood metabolites reflect the extent of recovery following SCI; (b) whether changes in blood-derived metabolites serve as prognostic indicators of patient outcomes based on the spinal cord independence measure (SCIM); and (c) whether metabolic pathways involved in recovery processes may provide insights into mechanisms that mediate neural damage and repair. Morning blood samples were collected from male complete and incomplete SCI patients (n = 7) following injury and at 6 months post-injury. Multivariate analyses were used to identify changes in serum metabolic profiles and were correlated to clinical outcomes. Specifically, acetyl phosphate, 1,3,7-trimethyluric acid, 1,9-dimethyluric acid, and acetic acid significantly related to SCIM scores. These preliminary findings suggest that specific metabolites may serve as proxy measures of the SCI phenotype and prognostic markers of recovery. Thus, serum metabolite analysis combined with machine learning holds promise in understanding the physiology of SCI and aiding in prognosticating outcomes following injury.