Characterization and mechanistic study of a radical SAM dehydrogenase in the biosynthesis of butirosin

BtrN encoded in the butirosin biosynthetic gene cluster possesses a CXXXCXXC motif conserved within the radical S-adenosyl methionine (SAM) superfamily. Its gene disruption in the butirosin producer Bacillus circulans caused the interruption of the biosynthetic pathway between 2-deoxy-scyllo-inosamine (DOIA) and 2-deoxystreptamine (DOS). Further, in vitro assay of the overexpressed enzyme revealed that BtrN catalyzed the oxidation of DOIA under the strictly anaerobic conditions along with consumption of an equimolar amount of SAM to produce 5'-deoxyadenosine, methionine, and 3-amino-2,3-dideoxy-scyllo-inosose (amino-DOI). Kinetic analysis showed substrate inhibition by DOIA but not by SAM, which suggests that the reaction is the Ordered Bi Ter mechanism and that SAM is the first substrate and DOIA is the second. The BtrN reaction with [3-2H]DOIA generated nonlabeled, monodeuterated and dideuterated 5'-deoxyadenosines, while no deuterium was incorporated by incubation of nonlabeled DOIA in the deuterium oxide buffer. These results indicated that the hydrogen atom at C-3 of DOIA was directly transferred to 5'-deoxyadenosine to give the radical intermediate of DOIA. Generation of nonlabeled and dideuterated 5'-deoxyadenosines proved the reversibility of the hydrogen abstraction step. The present study suggests that BtrN is an unusual radical SAM dehydrogenase catalyzing the oxidation of the hydroxyl group by a radical mechanism. This is the first report of the mechanistic study on the oxidation of a hydroxyl group by a radical SAM enzyme.     

C3′‐Deoxygenation of Paromamine Catalyzed by a Radical S‐Adenosylmethionine Enzyme: Characterization of the Enzyme AprD4 and Its Reductase Partner AprD3

C3′‐deoxygenation of aminoglycosides results in their decreased susceptibility to phosphorylation thereby increasing their efficacy as antibiotics. However, the biosynthetic mechanism of C3′‐deoxygenation is unknown. To address this issue, aprD4 and aprD3 genes from the apramycin gene cluster in Streptomyces tenebrarius were expressed in E. coli and the resulting gene products were characterized in vitro. AprD4 is shown to be a radical S‐adenosylmethionine (SAM) enzyme, catalyzing homolysis of SAM to 5′‐deoxyadenosine (5′‐dAdo) in the presence of paromamine. [4′‐²H]‐Paromamine was prepared and used to show that its C4′‐H is transferred to 5′‐dAdo by AprD4, during which the substrate is dehydrated to a product consistent with 4′‐oxolividamine. In contrast, paromamine is reduced to a deoxy product when incubated with AprD4/AprD3/NADPH. These results show that AprD4 is the first radical SAM diol‐dehydratase and, along with AprD3, is responsible for 3′‐deoxygenation in aminoglycoside biosynthesis.     

Characterisation of Streptomyces spheroides NovW and revision of its functional assignment to a dTDP-6-deoxy-D-xylo-4-hexulose 3-epimerase

Characterisation of recombinant Streptomyces spheroides NovW in vitro suggests that it is not a kinetically competent dual action dTDP-6-deoxy-d-xylo-4-hexulose 3,5-epimerase, but possesses only significant 3-epimerase activity.     

Biosynthesis of the Carbamoylated D‐Gulosamine Moiety of Streptothricins: Involvement of a Guanidino‐N‐glycosyltransferase and an N‐Acetyl‐D‐gulosamine Deacetylase

Streptothricins (STNs) are atypical aminoglycosides containing a rare carbamoylated D ‐gulosamine (D ‐GulN) moiety, and the antimicrobial activity of STNs has been exploited for crop protection. Herein, the biosynthetic pathway of the carbamoylated D ‐GulN moiety was delineated. An N‐acetyl‐D ‐galactosamine is first attached to the streptolidine lactam by the glycosyltransferse StnG and then epimerized to N‐acetyl‐D ‐gulosamine by the putative epimerase StnJ. After carbamoylation by the carbamoyltransferase StnQ, N‐acetyl‐D ‐GulN is deacetylated by StnI to furnish the carbamoylated D ‐GulN moiety. In vitro studies characterized two novel enzymes: StnG is an unprecedented GT‐A fold N‐glycosyltransferase that glycosylates the imine nitrogen atom of guanidine, and StnI is the first reported N‐acetyl‐D ‐GulN deacetylase.     

Purification and characterization of organic solvent stable serine alkaline protease from newly isolated Bacillus circulans M34

A protease from newly isolated Bacillus circulans M34 was purified by Q‐Sepharose anion exchange chromatography and Sepharose–bacitracin affinity chromatography followed by (NH₄)₂SO₄ precipitation. The molecular mass of the purified enzyme was determined using SDS–PAGE. The optimum pH and temperature for protease activity were 11 and 50°C, respectively. The effect of various metal ions on protease activity was investigated. Alkaline protease from Bacillus circulans M34 wase activated by Zn²⁺, Cu²⁺ and Co²⁺ up to 31%. The purified protease was found to be stable in the organic solvents, surfactants and oxidizing agent. The substrate specificity of purified protease was investigated towards different substrates. The protease was almost completely inhibited by the serine protease inhibitor phenylmethanesulfonyl fluoride. The kinetic parameters of the purified protease, maximum rate (V_max) and Michaelis constant (K_m), were determined using a Lineweaver–Burk plot. Copyright © 2015 John Wiley & Sons, Ltd.     

Characterization of 1,4-dihydroxy-2-naphthoyl-coenzyme A synthase (MenB) in phylloquinone biosynthesis of Synechocystis sp. PCC 6803

The gene product Sll1127 is a predicted 1,4-dihydroxy-2-naphthoyl-CoA synthase catalyzing an intramolecular Claisen condensation in the phylloquinone biosynthesis of the cyanobacterium Synechocystis sp.PCC 6803.This predicted catalytic function has been verified and the enzyme has been characterized for the first time with kcat = 0.013 s-1 and KM = 9μM.Its catalytic activity is found to strictly depend on externally added bicarbonate with an apparent KD = 0.60 mM.In addition,this enzyme is inhibited by its 1,4-dihydroxy-2-naphthoyl-CoA product through high-affinity binding,which causes a 18 nm shift of the inhibitor absorption at 392 to 410 nm and engenders a new absorption peak at 345 nm.All these properties of the cyanobacterial enzyme are closely similar to those of the Escherichia coli orthologue from the menaquinone biosynthetic pathway.These results provide additional supporting evidence for the essential role of bicarbonate as a catalytic base in the enzymatic reaction and the eubacterial origin of the enzymes in the cyanobacterial biosynthesis of phylloquinone.     

Purification and characterization of theD-hydantoinase from bacillus circulans

AD-hydantoinase (5,6-dihydropyrimidine amidohydrolase) was purified to homogeneity fromBacillus circulans. Purification of two hundred forty-three-fold was achieved with an overall yield of 12%. The relative molecular mass of the native enzyme is 212,000 and that of the subunit is 53,000. This enzyme is an acidic protein with an isoelectric point of 4.55. The enzyme is sensitive to thiol reagent and requires metal ions for its activity. The optimal conditions for the hydantoinase activity are pH 8.0–10.0 and a temperature of 75‡C. The enzyme is the most stable in a pH range of 8.5–9.5 and up to 60‡C. The enzyme is significantly stable not only at high temperatures but also on treatment with protein denaturant SDS. These remarkable properties are used for the purification procedure.     

Process optimisation for the biosynthesis of cellulase by Bacillus PC-BC6 and its mutant derivative Bacillus N3 using submerged fermentation

This study deals with optimisation of cultural conditions for enhanced production of cellulase by Bacillus PC-BC6 and its mutant derivative Bacillus N3. Influence of different variables including incubation time, temperature, inoculum size, pH, nitrogen sources and metal ions has been studied. The optimum conditions for cellulase production were incubation period of 72 h, inoculum size 4% incubation temperature 37°C, pH 7, 0.25% ammonium sulphate, 0.2% peptone as inorganic and organic nitrogen source in case of Bacillus PC-BC6. In case of mutant Bacillus N3, optimal conditions were incubation period of 48 h, incubation temperature 37°C, inoculum size 3%, pH 7, 0.2% ammonium chloride and 0.15% yeast extract. Presence of MnSO₄ and CaCl₂ enhances the enzyme production by Bacillus PC-BC6 and mutant Bacillus N3, respectively. This study was innovative and successful in producing cellulase economically by using cheap indigenous substrate Saccharum spontaneum.