Engineering Glucosamine-6-Phosphate Synthase to Achieve Efficient One-Step Biosynthesis of Glucosamine.

As an important functional monosaccharide, glucosamine (GlcN) is widely used in fields such as medicine, food nutrition, and health care. Here, we report a distinct GlcN biosynthesis method that utilizes engineered Bacillus subtilis glucosamine-6-phosphate synthase (BsGlmS) to convert D-fructose to directly generate GlcN. The best variant obtained by using a combinatorial active-site saturation test/iterative saturation mutagenesis (CAST/ISM) strategy was a quadruple mutant S596D/V597G/S347H/G299Q (BsGlmS-BK19), which has a catalytic activity 1736-fold that of the wild type toward D-fructose. Upon using mutant BK19 as a whole-cell catalyst, D-fructose was converted into GlcN with 65.32% conversion in 6 h, whereas the wild type only attained a conversion rate of 0.31% under the same conditions. Molecular docking and molecular dynamics simulations were implemented to provide insights into the mechanism underlying the enhanced activity of BK19. Importantly, the BsGlmS-BK19 variant specifically catalyzes D-fructose without the need for phosphorylated substrates, representing a significant advancement in GlcN biosynthesis.

Substrate Substitution in Kanosamine Biosynthesis Using Phosphonates and Phosphite Rescue.

Kanosamine is an antibiotic and antifungal compound synthesized from glucose 6-phosphate (G6P) in Bacillus subtilis by the action of three enzymes: NtdC, which catalyzes NAD-dependent oxidation of the C3-hydroxyl; NtdA, a PLP-dependent aminotransferase; and NtdB, a phosphatase. We previously demonstrated that NtdC can also oxidize substrates such as glucose and xylose, though at much lower rates, suggesting that the phosphoryloxymethylene moiety of the substrate is critical for effective catalysis. To probe this, we synthesized two phosphonate analogues of G6P in which the bridging oxygen is replaced by methylene and difluoromethylene groups. These analogues are substrates for NtdC, with second-order rate constants an order of magnitude lower than those for G6P. NtdA converts the resulting 3-keto products to the corresponding kanosamine 6-phosphonate analogues. We compared the rates to the rate of NtdC oxidation of glucose and xylose and showed that the low reactivity of xylose could be rescued 4-fold by the presence of phosphite, mimicking G6P in two pieces. These results allow the evaluation of the individual energetic contributions to catalysis of the bridging oxygen, the bridging C6 methylene, the phosphodianion, and the entropic gain of one substrate versus two substrate pieces. Phosphite also rescued the reversible formation 3-amino-3-deoxy-d-xylose by NtdA, demonstrating that truncated and nonhydrolyzable analogues of kanosamine 6-phosphate can be generated enzymatically.

Snapshots along the catalytic path of KabA, a PLP-dependent aminotransferase required for kanosamine biosynthesis in Bacillus cereus UW85.

Kanosamine is an antibiotic and antifungal monosaccharide. The kanosamine biosynthetic pathway from glucose 6-phosphate in Bacillus cereus UW85 was recently reported, and the functions of each of the three enzymes in the pathway, KabA, KabB and KabC, were demonstrated. KabA, a member of a subclass of the VIß family of PLP-dependent aminotransferases, catalyzes the second step in the pathway, generating kanosamine 6-phosphate (K6P) using l-glutamate as the amino-donor. KabA catalysis was shown to be extremely efficient, with a second-order rate constant with respect to K6P transamination of over 107 M-1s-1. Here we report the high-resolution structure of KabA in both the PLP- and PMP-bound forms. In addition, co-crystallization with K6P allowed the structure of KabA in complex with the covalent PLP-K6P adduct to be solved. Co-crystallization or soaking with glutamate or 2-oxoglutarate did not result in crystals with either substrate/product. Reduction of the PLP-KabA complex with sodium cyanoborohydride gave an inactivated enzyme, and crystals of the reduced KabA were soaked with the l-glutamate analog glutarate to mimic the KabA-PLP-l-glutamate complex. Together these four structures give a complete picture of how the active site of KabA recognizes substrates for each half-reaction. The KabA structure is discussed in the context of homologous aminotransferases.

Genome analysis of 1-deoxynojirimycin (1-DNJ)-producing Bacillus velezensis K26 and distribution of Bacillus sp. harboring a 1-DNJ biosynthetic gene cluster.

1-Deoxynojirumycin (1-DNJ) is a representative iminosugar with α-glucosidase inhibition (AGI) activity. In this study, the full genome sequencing of 1-DNJ-producing Bacillus velezensis K26 was performed. The genome consists of a circular chromosome (4,047,350 bps) with two types of putative virulence factors, five antibiotic resistance genes, and seven secondary metabolite biosynthetic gene clusters. Genomic analysis of a wide range of Bacillus species revealed that a 1-DNJ biosynthetic gene cluster was commonly present in four Bacillus species (B. velezensis, B. pseudomycoides, B. amyloliquefaciens, and B. atrophaeus). In vitro experiments revealed that the increased mRNA expression levels of the three 1-DNJ biosynthetic genes were closely related to increased AGI activity. Genomic comparison and alignment of multiple gene sequences indicated the conservation of the 1-DNJ biosynthetic gene cluster in each Bacillus species. This genomic analysis of Bacillus species having a 1-DNJ biosynthetic gene cluster could provide a basis for further research on 1-DNJ-producing bacteria.

Kinetic Study of Glucosamine Production Using Aspergillus sydowii BCRC 31742 under Solid-State Fermentation.

In the present study, we aimed to obtain a high yield and productivity for glucosamine using a low-cost solid-state culture with Aspergillus sydowii BCRC 31742. The fermentation conditions, such as inoculum biomass, moisture content, and supplemental volume and mineral salt, were chosen to achieve high productivity of glucosamine (GlcN). When the initial supplemental volume used was 3 mL/g substrate, the yield and productivity of GlcN were 48.7 mg/gds and 0.69 mg/gds·h, respectively. This result will be helpful for the industrialization of the process.

Carbocyclic Substrate Analogues Reveal Kanosamine Biosynthesis Begins with the α-Anomer of Glucose 6-Phosphate.

NtdC is an NAD-dependent dehydrogenase that catalyzes the conversion of glucose 6-phosphate (G6P) to 3-oxo-glucose 6-phosphate (3oG6P), the first step in kanosamine biosynthesis in Bacillus subtilis and other closely-related bacteria. The NtdC-catalyzed reaction is unusual because 3oG6P undergoes rapid ring opening, resulting in a 1,3-dicarbonyl compound that is inherently unstable due to enolate formation. We have reported the steady-state kinetic behavior of NtdC, but many questions remain about the nature of this reaction, including whether it is the α-anomer, ß-anomer, or open-chain form that is the substrate for the enzyme. Here, we report the synthesis of carbocyclic G6P analogues by two routes, one based upon the Ferrier II rearrangement to generate the carbocycle and one based upon a Claisen rearrangement. We were able to synthesize both pseudo-anomers of carbaglucose 6-phosphate (C6P) using the Ferrier approach, and activity assays revealed that the pseudo-α-anomer is a good substrate for NtdC, while the pseudo-ß-anomer and the open-chain analogue, sorbitol 6-phosphate (S6P), are not substrates. A more efficient synthesis of α-C6P was achieved using the Claisen rearrangement approach, which allowed for a thorough evaluation of the NtdC-catalyzed oxidation of α-C6P. The requirement for the α-anomer indicates that NtdC and NtdA, the subsequent enzyme in the pathway, have co-evolved to recognize the α-anomer in order to avoid mutarotation between enzymatic steps.

Glucosamine-P and rhodococcal ADP-glucose pyrophosphorylases: A hint to (re)discover (actino)bacterial amino sugar metabolism.

Glycogen was described as a temporal storage molecule in rhodococci, interconnecting lipids and carbon availability. The Rhodococcus jostii ADP-glucose pyrophosphorylase (ADP-GlcPPase) kinetic and regulatory properties support this role. Curiously, the enzyme uses glucosamine-1P as alternative substrate. Herein, we report the in-depth study of glucosamine-1P activity and its regulation in two rhodocoocal ADP-GlcPPases, finding that glucosamine-6P (representing a metabolic carbon/nitrogen node) is a critical activator, then reinforcing the role of glycogen as an "intermediary metabolite" in rhodococci. Glucosamine-1P activity in rhodococcal ADP-GlcPPases responds to activation by metabolites improving their catalytic performance, strongly suggesting its metabolic feasibility. This work supports a scenario for new molecules/metabolites discovery and hypothesizes on evolutionary mechanisms underlying enzyme promiscuity opening novel metabolic features in (actino)bacteria.

Sustainable and Effective Chitosan Production by Dimorphic Fungus Mucor rouxii via Replacing Yeast Extract with Fungal Extract.

The effects of switching morphology and replacing supplementary nutrients with fungal extract (5 and 10 g/L) on the production of major metabolites and chitosan by Mucor rouxii were investigated. This approach was supposed to promote sustainability of the fermentation process and improve its economic feasibility. Different fungal morphologies, i.e., purely filamentous (PF), purely yeast-like (PY), mostly filamentous (MF), and mostly yeast-like (MY), were evaluated. The highest ethanol yields were obtained from the media supplemented with 10 g/L fungal extract for all morphologies, while adding nutrient salts did not make any improvements in these yields, except a slight decrease in the fermentation time. Except for PF morphology, the replacement of yeast extract favored the biomass production yields. Moreover, the alkali insoluble material (AIM) yields were higher as a result of the replacement for most cases. Furthermore, the replacement resulted in increased glucosamine and decreased N-acetyl-glucosamine content of AIM for almost all the morphologies. AIM yields of at least 0.25 g/g-glucose and maximum chitin/chitosan yield of 0.78 g/g-AIM were obtained from the solids remaining after autolysis process, which were higher than that obtained from the raw biomass. The maximum yield of 0.135 g/g-AIM purified chitosan with intact molecular weight was obtained from the biomass with PF morphology supplemented with 10 g/L fungal extract plus nutrients.

X-ray crystallographic structure of BshB, the zinc-dependent deacetylase involved in bacillithiol biosynthesis.

Many gram-positive bacteria produce bacillithiol to aid in the maintenance of redox homeostasis and degradation of toxic compounds, including the antibiotic fosfomycin. Bacillithiol is produced via a three-enzyme pathway that includes the action of the zinc-dependent deacetylase BshB. Previous studies identified conserved aspartate and histidine residues within the active site that are involved in metal binding and catalysis, but the enzymatic mechanism is not fully understood. Here we report two X-ray crystallographic structures of BshB from Bacillus subtilis that provide insight into the BshB catalytic mechanism.

The kanosamine biosynthetic pathway in Bacillus cereus UW85: Functional and kinetic characterization of KabA, KabB, and KabC.

Kanosamine is an aminosugar antibiotic, and component of complex antibiotics such as kanamycin. The biosynthesis of kanosamine varies among different bacteria; best known is a pathway starting from UDP-glucose, but Bacillus subtilis can produce kanosamine in a three-step pathway from glucose 6-phosphate. A set of genes proposed to encode a kanosamine pathway has previously been identified within the zwittermicin A gene cluster of Bacillus cereus UW85. These genes, designated kabABC, are similar to the B. subtilis kanosamine pathway genes (ntdABC), but have never been studied experimentally. We have expressed each of the kab genes, and studied the in vitro substrate scope and reaction rates and kinetic mechanisms of all three enzymes. The kab genes encode enzymes that catalyze a route similar to that found in B. subtilis from glucose 6-phosphate to kanosamine, passing through an unusual and unstable 3-keto intermediate. Kinetic studies show the first step in the pathway, the KabC-catalyzed oxidation of glucose 6-phosphate at carbon-3, is very slow relative to the subsequent KabA-catalyzed aminotransferase and KabB-catalyzed phosphatase reactions. KabC differs from its homolog, NtdC, in that it is NADP- rather than NAD-dependent. The KabA kinetic study is the first such report for a kanosamine 6-phosphate aminotransferase, revealing an extremely efficient PLP-dependent reaction. These results show that this kanosamine biosynthesis pathway occurs in more than one organism, and that the reactions are tuned in order to avoid any accumulation of the unstable intermediate.

O-GlcNAcylation-dependent upregulation of HO1 triggers ammonia-induced oxidative stress and senescence in hepatic encephalopathy.

BACKGROUND & AIMS: Cerebral oxidative stress plays an important role in the pathogenesis of hepatic encephalopathy (HE), but the underlying mechanisms are incompletely understood. Herein, we analyzed a role of heme oxygenase (HO)1, iron and NADPH oxidase 4 (Nox4) for the induction of oxidative stress and senescence in HE. METHODS: Gene and protein expression in human post-mortem brain samples was analyzed by gene array and western blot analysis. Mechanisms and functional consequences of HO1 upregulation were studied in NH4Cl-exposed astrocytes in vitro by western blot, qPCR and super-resolution microscopy. RESULTS: HO1 and the endoplasmic reticulum (ER) stress marker grp78 were upregulated, together with changes in the expression of multiple iron metabolism-related genes, in post-mortem brain samples from patients with liver cirrhosis and HE. NH4Cl elevated HO1 protein and mRNA in cultured astrocytes through glutamine synthetase (GS)-dependent upregulation of glutamine/fructose amidotransferases 1/2 (GFAT1/2), which blocked the transcription of the HO1-targeting miR326-3p in a O-GlcNAcylation dependent manner. Upregulation of HO1 by NH4Cl triggered ER stress and was associated with elevated levels of free ferrous iron and expression changes in iron metabolism-related genes, which were largely abolished after knockdown or inhibition of GS, GFAT1/2, HO1 or iron chelation. NH4Cl, glucosamine (GlcN) and inhibition of miR326-3p upregulated Nox4, while knockdown of Nox4, GS, GFAT1/2, HO1 or iron chelation prevented NH4Cl-induced RNA oxidation and astrocyte senescence. Elevated levels of grp78 and O-GlcNAcylated proteins were also found in brain samples from patients with liver cirrhosis and HE. CONCLUSION: The present study identified glucosamine synthesis-dependent protein O-GlcNAcylation as a novel mechanism in the pathogenesis of HE that triggers oxidative and ER stress, as well as senescence, through upregulation of HO1 and Nox4. LAY SUMMARY: Patients with liver cirrhosis frequently exhibit hyperammonemia and suffer from cognitive and motoric dysfunctions, which at least in part involve premature ageing of the astrocytes in the brain. This study identifies glucosamine and an O-GlcNAcylation-dependent disruption of iron homeostasis as novel triggers of oxidative stress, thereby mediating ammonia toxicity in the brain.

A structural and functional analysis of the glycosyltransferase BshA from Staphylococcus aureus: Insights into the reaction mechanism and regulation of bacillithiol production.

Bacillithiol is a glucosamine-derived antioxidant found in several pathogenic Gram-positive bacteria. The compound is involved in maintaining the appropriate redox state within the cell as well as detoxifying foreign agents like the antibiotic fosfomycin. Bacillithiol is produced via the action of three enzymes, including BshA, a retaining GT-B glycosyltransferase that utilizes UDP-N-acetylglucosamine and l-malate to produce N-acetylglucosaminyl-malate. Recent studies suggest that retaining GT-B glycosyltransferases like BshA utilize a substrate-assisted mechanism that goes through an SN i-like transition state. In a previous study, we relied on X-ray crystallography as well as computational simulations to hypothesize the manner in which substrates would bind the enzyme, but several questions about substrate binding and the role of one of the amino acid residues persisted. Another study demonstrated that BshA might be subject to feedback inhibition by bacillithiol, but this phenomenon was not analyzed further to determine the exact mechanism of inhibition. Here we present X-ray crystallographic structures and steady-state kinetics results that help elucidate both of these issues. Our ligand-bound crystal structures demonstrate that the active site provides an appropriate steric and geometric arrangement of ligands to facilitate the substrate-assisted mechanism. Finally, we show that bacillithiol is competitive for UDP-N-acetylglucosamine with a Ki value near 120-130 µM and likely binds within the BshA active site, suggesting that bacillithiol modulates BshA activity via feedback inhibition. The work presented here furthers our understanding of bacillithiol metabolism and can aid in the development of inhibitors to counteract resistance to antibiotics such as fosfomycin.

Mammalian Target of Rapamycin 2 (MTOR2) and C-MYC Modulate Glucosamine-6-Phosphate Synthesis in Glioblastoma (GBM) Cells Through Glutamine: Fructose-6-Phosphate Aminotransferase 1 (GFAT1).

Glucose and glutamine are two essential ingredients for cell growth. Glycolysis and glutaminolysis can be linked by glutamine: fructose-6-phosphate aminotransferase (GFAT, composed of GFAT1 and GFAT2) that catalyzes the synthesis of glucosamine-6-phosphate and glutamate by using fructose-6-phosphate and glutamine as substrates. The role of mammalian target of rapamycin (MTOR, composed of MTOR1 and MTOR2) in regulating glycolysis has been explored in human cancer cells. However, whether MTOR can interact with GFAT to regulate glucosamine-6-phosphate is poorly understood. In this study, we report that GFAT1 is essential to maintain the malignant features of GBM cells. And MTOR2 rather than MTOR1 plays a robust role in promoting GFAT1 protein activity, and accelerating the progression of glucosamine-6-phosphate synthesis, which is not controlled by the PI3K/AKT signaling. Intriguingly, high level of glucose or glutamine supply promotes MTOR2 protein activity. In turn, up-regulating glycolytic and glutaminolytic metabolisms block MTOR dimerization, enhancing the release of MTOR2 from the MTOR complex. As a transcriptional factor, C-MYC, directly targeted by MTOR2, promotes the relative mRNA expression level of GFAT1. Notably, our data reveal that GFAT1 immunoreactivity is positively correlated with the malignant grades of glioma patients. Kaplan-Meier assay reveals the correlations between patients' 5-year survival and high GFAT1 protein expression. Taken together, we propose that the MTOR2/C-MYC/GFAT1 axis is responsible for the modulation on the crosstalk between glycolysis and glutaminolysis in GBM cells. Under the condition of accelerated glycolytic and/or glutaminolytic metabolisms, the MTOR2/C-MYC/GFAT1 axis will be up-regulated in GBM cells.

Regulated Assembly of LPS, Its Structural Alterations and Cellular Response to LPS Defects.

Distinguishing feature of the outer membrane (OM) of Gram-negative bacteria is its asymmetry due to the presence of lipopolysaccharide (LPS) in the outer leaflet of the OM and phospholipids in the inner leaflet. Recent studies have revealed the existence of regulatory controls that ensure a balanced biosynthesis of LPS and phospholipids, both of which are essential for bacterial viability. LPS provides the essential permeability barrier function and act as a major virulence determinant. In Escherichia coli, more than 100 genes are required for LPS synthesis, its assembly at inner leaflet of the inner membrane (IM), extraction from the IM, translocation to the OM, and in its structural alterations in response to various environmental and stress signals. Although LPS are highly heterogeneous, they share common structural elements defining their most conserved hydrophobic lipid A part to which a core polysaccharide is attached, which is further extended in smooth bacteria by O-antigen. Defects or any imbalance in LPS biosynthesis cause major cellular defects, which elicit envelope responsive signal transduction controlled by RpoE sigma factor and two-component systems (TCS). RpoE regulon members and specific TCSs, including their non-coding arm, regulate incorporation of non-stoichiometric modifications of LPS, contributing to LPS heterogeneity and impacting antibiotic resistance.

Redox regulation by reversible protein S-thiolation in Gram-positive bacteria.

Low molecular weight (LMW) thiols play an important role as thiol-cofactors for many enzymes and are crucial to maintain the reduced state of the cytoplasm. Most Gram-negative bacteria utilize glutathione (GSH) as major LMW thiol. However, in Gram-positive Actinomycetes and Firmicutes alternative LMW thiols, such as mycothiol (MSH) and bacillithiol (BSH) play related roles as GSH surrogates, respectively. Under conditions of hypochlorite stress, MSH and BSH are known to form mixed disulfides with protein thiols, termed as S-mycothiolation or S-bacillithiolation that function in thiol-protection and redox regulation. Protein S-thiolations are widespread redox-modifications discovered in different Gram-positive bacteria, such as Bacillus and Staphylococcus species, Mycobacterium smegmatis, Corynebacterium glutamicum and Corynebacterium diphtheriae. S-thiolated proteins are mainly involved in cellular metabolism, protein translation, redox regulation and antioxidant functions with some conserved targets across bacteria. The reduction of protein S-mycothiolations and S-bacillithiolations requires glutaredoxin-related mycoredoxin and bacilliredoxin pathways to regenerate protein functions. In this review, we present an overview of the functions of mycothiol and bacillithiol and their physiological roles in protein S-bacillithiolations and S-mycothiolations in Gram-positive bacteria. Significant progress has been made to characterize the role of protein S-thiolation in redox-regulation and thiol protection of main metabolic and antioxidant enzymes. However, the physiological roles of the pathways for regeneration are only beginning to emerge as well as their interactions with other cellular redox systems. Future studies should be also directed to explore the roles of protein S-thiolations and their redox pathways in pathogenic bacteria under infection conditions to discover new drug targets and treatment options against multiple antibiotic resistant bacteria.

Biocatalytic Production of Glucosamine from N-Acetylglucosamine by Diacetylchitobiose Deacetylase.

Glucosamine (GlcN) is widely used in the nutraceutical and pharmaceutical industries. Currently, GlcN is mainly produced by traditional multistep chemical synthesis and acid hydrolysis, which can cause severe environmental pollution, require a long prodution period but a lower yield. The aim of this work was to develop a whole-cell biocatalytic process for the environment-friendly synthesis of glucosamine (GlcN) from N-acetylglucosamine (GlcNAc). We constructed a recombinant Escherichia coli and Bacillus subtilis strains as efficient whole-cell biocatalysts via expression of diacetylchitobiose deacetylase (Dacph) from Pyrococcus furiosus. Although both strains were biocatalytically active, the performance of B. subtilis was better. To enhance GlcN production, optimal reaction conditions were found: B. subtilis whole-cell biocatalyst 18.6 g/l, temperature 40°C, pH 7.5, GlcNAc concentration 50 g/l and reaction time 3 h. Under the above conditions, the maximal titer of GlcN was 35.3 g/l, the molar conversion ratio was 86.8% in 3-L bioreactor. This paper shows an efficient biotransformation process for the biotechnological production of GlcN in B. subtilis that is more environmentally friendly than the traditional multistep chemical synthesis approach. The biocatalytic process described here has the advantage of less environmental pollution and thus has great potential for large-scale production of GlcN in an environment-friendly manner.

The Mycobacterium tuberculosis complex has a pathway for the biosynthesis of 4-formamido-4,6-dideoxy-d-glucose.

Recent studies have demonstrated that the O-antigens of some pathogenic bacteria such as Brucella abortus, Francisella tularensis, and Campylobacter jejuni contain quite unusual N-formylated sugars (3-formamido-3,6-dideoxy-d-glucose or 4-formamido-4,6-dideoxy-d-glucose). Typically, four enzymes are required for the formation of such sugars: a thymidylyltransferase, a 4,6-dehydratase, a pyridoxal 5'-phosphate or PLP-dependent aminotransferase, and an N-formyltransferase. To date, there have been no published reports of N-formylated sugars associated with Mycobacterium tuberculosis. A recent investigation from our laboratories, however, has demonstrated that one gene product from M. tuberculosis, Rv3404c, functions as a sugar N-formyltransferase. Given that M. tuberculosis produces l-rhamnose, both a thymidylyltransferase (Rv0334) and a 4,6-dehydratase (Rv3464) required for its formation have been identified. Thus, there is one remaining enzyme needed for the production of an N-formylated sugar in M. tuberculosis, namely a PLP-dependent aminotransferase. Here we demonstrate that the M. tuberculosis rv3402c gene encodes such an enzyme. Our data prove that M. tuberculosis contains all of the enzymatic activities required for the formation of dTDP-4-formamido-4,6-dideoxy-d-glucose. Indeed, the rv3402c gene product likely contributes to virulence or persistence during infection, though its temporal expression and location remain to be determined.

The aldehyde dehydrogenase AldA contributes to the hypochlorite defense and is redox-controlled by protein S-bacillithiolation in Staphylococcus aureus.

Staphylococcus aureus produces bacillithiol (BSH) as major low molecular weight (LMW) thiol which functions in thiol-protection and redox-regulation by protein S-bacillithiolation under hypochlorite stress. The aldehyde dehydrogenase AldA was identified as S-bacillithiolated at its active site Cys279 under NaOCl stress in S. aureus. Here, we have studied the expression, function, redox regulation and structural changes of AldA of S. aureus. Transcription of aldA was previously shown to be regulated by the alternative sigma factor SigmaB. Northern blot analysis revealed SigmaB-independent induction of aldA transcription under formaldehyde, methylglyoxal, diamide and NaOCl stress. Deletion of aldA resulted in a NaOCl-sensitive phenotype in survival assays, suggesting an important role of AldA in the NaOCl stress defense. Purified AldA showed broad substrate specificity for oxidation of several aldehydes, including formaldehyde, methylglyoxal, acetaldehyde and glycol aldehyde. Thus, AldA could be involved in detoxification of aldehyde substrates that are elevated under NaOCl stress. Kinetic activity assays revealed that AldA is irreversibly inhibited under H2O2 treatment in vitro due to overoxidation of Cys279 in the absence of BSH. Pre-treatment of AldA with BSH prior to H2O2 exposure resulted in reversible AldA inactivation due to S-bacillithiolation as revealed by activity assays and BSH-specific Western blot analysis. Using molecular docking and molecular dynamic simulation, we further show that BSH occupies two different positions in the AldA active site depending on the AldA activation state. In conclusion, we show here that AldA is an important target for S-bacillithiolation in S. aureus that is up-regulated under NaOCl stress and functions in protection under hypochlorite stress.

Simultaneous Measurement of Glucose-6-phosphate 3-Dehydrogenase (NtdC) Catalysis and the Nonenzymatic Reaction of Its Product: Kinetics and Isotope Effects on the First Step in Kanosamine Biosynthesis.

Glucose-6-phosphate 3-dehydrogenase (NtdC) is an NAD-dependent oxidoreductase encoded in the NTD operon of Bacillus subtilis. The oxidation of glucose 6-phosphate by NtdC is the first step in kanosamine biosynthesis. The product, 3-oxo-d-glucose 6-phosphate (3oG6P), has never been synthesized or isolated. The NtdC-catalyzed reaction is very slow at low and neutral pH, and its rate increases to a maximum near pH 9.5. However, under alkaline conditions, the product is not stable because of ring opening followed by deprotonation of the 1,3-dicarbonyl compound. The absorbance band due to this enolate at 310 nm overlaps with that of the other enzymatic product, NADH, complicating kinetic measurements. We report the deconvolution of the resulting spectra of the reaction to determine the rate constants and likely kinetic mechanism. In doing so, we were able to determine the extinction coefficient of the enolate of 3oG6P (23000 M-1 cm-1), which allowed the measurement of the first-order rate constant (5.51 × 10-3 s-1) and activation energy (93 kJ mol-1) of nonenzymatic enolate formation. Using deuterium-labeled substrates, we show that hydride transfer from carbon 3 is partially rate-limiting in the enzymatic reaction, and deuterium substitution on carbon 2 has no significant effect on the enzymatic reaction but lowers the rate of deprotonation of 3oG6P 4-fold. These experiments clearly establish the regiochemistry of the reactions. Coupling of the NtdC reaction with the subsequent step in the pathway, NtdA-catalyzed glutamate-dependent amino transfer, has a small but significant effect on the rate of NAD reduction, consistent with these enzymes working together to process the unstable metabolite.

Critical Minireview: The Fate of tRNACys during Oxidative Stress in Bacillus subtilis.

Oxidative stress occurs when cells are exposed to elevated levels of reactive oxygen species that can damage biological molecules. One bacterial response to oxidative stress involves disulfide bond formation either between protein thiols or between protein thiols and low-molecular-weight (LMW) thiols. Bacillithiol was recently identified as a major low-molecular-weight thiol in Bacillus subtilis and related Firmicutes. Four genes (bshA, bshB1, bshB2, and bshC) are involved in bacillithiol biosynthesis. The bshA and bshB1 genes are part of a seven-gene operon (ypjD), which includes the essential gene cca, encoding CCA-tRNA nucleotidyltransferase. The inclusion of cca in the operon containing bacillithiol biosynthetic genes suggests that the integrity of the 3' terminus of tRNAs may also be important in oxidative stress. The addition of the 3' terminal CCA sequence by CCA-tRNA nucleotidyltransferase to give rise to a mature tRNA and functional molecules ready for aminoacylation plays an essential role during translation and expression of the genetic code. Any defects in these processes, such as the accumulation of shorter and defective tRNAs under oxidative stress, might exert a deleterious effect on cells. This review summarizes the physiological link between tRNACys regulation and oxidative stress in Bacillus.