A Fold Type II PLP-Dependent Enzyme from Fusobacterium nucleatum Functions as a Serine Synthase and Cysteine Synthase

Biochemistry ◽  
2021 ◽  
Vol 60 (7) ◽  
pp. 524-536
Author(s):  
Amanda L. Darbyshire ◽  
Robert G. Mothersole ◽  
Kirsten R. Wolthers
Keyword(s):  
Fusobacterium Nucleatum ◽  
Type Ii ◽  
Cysteine Synthase ◽  
Fold Type

scholarly journals Structural studies on the catalytic mechanism of Diaminopropionate ammonia lyase

2014 ◽  
Vol 70 (a1) ◽  
pp. C1822-C1822
Author(s):  
Geeta Deka ◽  
Shveta Bisht ◽  
H.S. Savithri ◽  
M.R.N Murthy
Keyword(s):  
Reaction Mechanism ◽  
Disulfide Bond ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Biochemical Characterization ◽  
Catalytic Efficiency ◽  
Peptide Antibiotics ◽  
Type Ii ◽  
Structural Studies ◽  
Fold Type

Diaminopropionate ammonia lyase (DAPAL) is a non-stereo specific fold-type II pyridoxal 5' phosphate (PLP) dependent enzyme that catalyzes the conversion of both D/L isoforms of the nonstandard amino acid Diaminopropionate (DAP) to pyruvate and ammonia. DAP is important for the synthesis of nonribosomal peptide antibiotics such as viomycin and capreomycin. Earlier structural studies on EcDAPAL bound to a reaction intermediate (aminoacrylate) suggested that the enzyme follows a two base mechanism, where Asp120 and Lys77 function as general bases to abstract proton from D-DAP and L-DAP respectively. A novel disulfide was observed near the active site, although its functional significance was not clear. In the present study, structural and biochemical characterization of active site mutants Asp120 (Asp120Asn/Ser/Thr/Cys) and Lys77 (Lys77His/ Thr/Ala/Val) of EcDAPAL has been carried out. Reduction of catalytic efficiency (Kcat/Km) of D120N EcDAPAL for D-DAP by 140 fold and presence of the uncatalyzed ligand at the active site in the crystal structure suggested that Asp120 indeed abstracts proton from D-DAP. Lys77, which was speculated to be important for proton abstraction from L DAP, however seemed to be crucial for PLP binding only. Presence of non-covalently bound PLP in the L77W mutant and occurence of both the ketoenamine, enolimine forms of internal aldimine in L77R mutant provided an in depth insight into the unique chemistry of internal aldimine formation in PLP dependent enzymes. To investigate the role of the novel disulfide bond near the active site, C265 and C291 were mutated to Serine. Studies on these mutants show that this disulfide bond gives additional stability to the protein and might regulate the entry of substrates to the active site. Thus, these studies provide deeper insights into the reaction mechanism of EcDAPAL, representing the overall reaction mechanism followed by several other fold-type II PLP pendent enzymes.

scholarly journals Identification and enzymic analysis of a novel protein associated with production of hydrogen sulfide and l-serine from l-cysteine in Fusobacterium nucleatum subsp. nucleatum ATCC 25586

Microbiology ◽  
2011 ◽  
Vol 157 (7) ◽  
pp. 2164-2171 ◽  
Author(s):  
Yasuo Yoshida ◽  
Kyosuke Suwabe ◽  
Keiji Nagano ◽  
Yuichiro Kezuka ◽  
Hirohisa Kato ◽  
...  
Keyword(s):  
Hydrogen Sulfide ◽  
Genomic Dna ◽  
Fusobacterium Nucleatum ◽  
Cosmid Library ◽  
Reverse Phase Hplc ◽  
Reverse Phase ◽  
Cysteine Synthase ◽  
Production Of Hydrogen ◽  
Novel Protein

A third enzyme that produces hydrogen sulfide from l-cysteine was identified in Fusobacterium nucleatum subsp. nucleatum. The fn1055 gene was cloned from a cosmid library constructed with genomic DNA of F. nucleatum ATCC 25586. Despite the database annotation that the product of fn1055 is a cysteine synthase, reverse-phase HPLC revealed that no l-cysteine was produced in vitro by the purified Fn1055 protein; however, the enzyme did produce l-serine. In addition, a cysteine auxotroph, Escherichia coli NK3, transformed with a plasmid containing the fn1055 gene did not grow without cysteine, which further suggests that Fn1055 does not function as a cysteine synthase. The Michaelis–Menten kinetics (K m = 0.09±0.001 mM and k cat = 5.43±0.64 s−1) of the purified enzyme showed that the capacity of Fn1055 to produce hydrogen sulfide was between that of two other enzymes, Fn0625 and Fn1220. Incubation of Fn1055 with l-cysteine resulted in the production of hydrogen sulfide, but not of pyruvate, ammonia or lanthionine, which are all byproducts produced in addition to hydrogen sulfide when Fn0625 or Fn1220 is incubated with l-cysteine. Instead, Fn1055 produced l-serine in its reaction with l-cysteine. Fn1055 produces hydrogen sulfide from l-cysteine by a mechanism that is different from that of Fn0625 or Fn1220.

Phonatory compensation in patients with larynx cancer after CO2 laser cordectomy

2019 ◽  
Vol 73 (6) ◽  
pp. 1-5
Author(s):  
Bożena Kosztyła-Hojna ◽  
Jarosław Łuczaj ◽  
Greta Berger ◽  
Emilia Duchnowska ◽  
Maciej Zdrojkowski ◽  
...  
Keyword(s):  
Cancer Treatment ◽  
Co2 Laser ◽  
Anterior Commissure ◽  
Type I ◽  
Type Ii ◽  
Type Iii ◽  
Type Iv ◽  
Fold Type ◽  
Compensation Mechanisms ◽  
Laser Cordectomy

Introduction: CO2 laser endoscopic cordectomy is the method of laryngeal cancer treatment. The type of cordectomy (I–VI) depends on the extent of the tumor. Endoscopic laser surgery provides more satisfactory phonation conditions in comparison to open surgical procedures. The aim of the study was to classify phonatory compensation mechanisms after CO2 laser cordectomy using the HSDI. Material and methods: The study included 30 men treated and diagnosed at the Department of Otolaryngology and Department of Clinical Phonoaudiology and Logopedics, Medical University of Bialystok. The control included 30 men with no pathological changes in the larynx. Type III, IV and Va CO2 laser cordectomy have been for glottis cancer treatment. Postoperative evaluation has been conducted 6 months after the surgery. HSDI has been used in larynx visualization. Results: Type I compensation occurs most frequently in patients after type III cordectomy. Advanced glottis cancer, as an indication for type IV and V cordectomy, leads to epiglottic hyperfunction and phonation involving vestibular folds – type II and III compensation. Type IV compensation is most frequent in type IV cordectomy. Conclusions: The type compensation is connected with the extent of glottis resection. In cordectomy including anterior commissure and the part of opposite fold (type Va), supraglottic hyperfunction with the participation of vestibular folds (type II and III compensation) has been recorded. Transmuscular cordectomy (type III) most often resulted in type I compensation. Type III-Va cordectomy caused reduction or absence of MW, decrease in amplitude and aperiodicity of vibrations in HSDI.

FOLD TYPE II PYRIDOXAL 5'-PHOSPHATE DEPENDENT ENZYMES: STRUCTURE, SUBSTRATE RECOGNITION AND CATALYSIS

2013 ◽  
pp. 398-416
Author(s):  
S. BISHT ◽  
S. R. BHARATH ◽  
H. S. SAVITHRI ◽  
M. R. N. MURTHY
Keyword(s):  
Substrate Recognition ◽  
Type Ii ◽  
Fold Type

Type II Solar Radio Bursts over Solar Cycle 21

1994 ◽  
Vol 144 ◽  
pp. 283-284
Author(s):  
G. Maris ◽  
E. Tifrea
Keyword(s):  
Solar Radio ◽  
Interplanetary Space ◽  
Impulsive Phase ◽  
Radio Bursts ◽  
Type Ii ◽  
Solar Radio Bursts ◽  
Strong Energy ◽  
Mass Ejection ◽  
Geophysical Effect ◽  
Radio Event

The type II solar radio bursts produced by a shock wave passing through the solar corona are one of the most frequently studied solar activity phenomena. The scientific interest in this type of phenomenon is due to the fact that the presence of this radio event in a solar flare is an almost certain indicator of a future geophysical effect. The origin of the shock waves which produce these bursts is not at all simple; besides the shocks which are generated as a result of a strong energy release during the impulsive phase of a flare, there are also the shocks generated by a coronal mass ejection or the shocks which appear in the interplanetary space due to the supplementary acceleration of the solar particles.

Heat-Etching with a Bullivant Type II Simple Freeze-Cleave Device

1970 ◽  
Vol 28 ◽  
pp. 106-107 ◽  
Author(s):  
Ronald S. Weinstein ◽  
N. Scott McNutt
Keyword(s):  
New Zealand ◽  
General Hospital ◽  
Massachusetts General Hospital ◽  
Type I ◽  
Type Ii ◽  
Collaborative Effort ◽  
Temperature And Pressure ◽  
The University

The Type I simple cold block device was described by Bullivant and Ames in 1966 and represented the product of the first successful effort to simplify the equipment required to do sophisticated freeze-cleave techniques. Bullivant, Weinstein and Someda described the Type II device which is a modification of the Type I device and was developed as a collaborative effort at the Massachusetts General Hospital and the University of Auckland, New Zealand. The modifications reduced specimen contamination and provided controlled specimen warming for heat-etching of fracture faces. We have now tested the Mass. General Hospital version of the Type II device (called the “Type II-MGH device”) on a wide variety of biological specimens and have established temperature and pressure curves for routine heat-etching with the device.

Ultrastructure of pulmonary microvasculature in acute endotoxemia

1978 ◽  
Vol 36 (2) ◽  
pp. 470-471
Author(s):  
R. G. Gerrity ◽  
M. Richardson
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Alveolar Epithelium ◽  
Endothelial Damage ◽  
Capillary Endothelium ◽  
Type Ii Cells ◽  
Type Ii ◽  
Interstitial Edema ◽  
Pulmonary Capillaries ◽  
Lung Ultrastructure ◽  
Fibrin Thrombi

Dogs were injected intravenously with E_. coli endotoxin (2 mg/kg), and lung samples were taken at 15 min., 1 hr. and 24 hrs. At 15 min., occlusion of pulmonary capillaries by degranulating platelets and polymorphonuclear leukocytes (PML) was evident (Fig. 1). Capillary endothelium was intact but endothelial damage in small arteries and arterioles, accompanied by intraalveolar hemorrhage, was frequent (Fig. 2). Sloughing of the surfactant layer from alveolar epithelium was evident (Fig. 1). At 1 hr., platelet-PML plugs were no longer seen in capillaries, the endothelium of which was often vacuolated (Fig. 3). Interstitial edema and destruction of alveolar epithelium were seen, and type II cells had discharged their granules into the alveoli (Fig. 4). At 24 hr. phagocytic PML's were frequent in peripheral alveoli, while centrally, alveoli and vessels were packed with fibrin thrombi and PML's (Fig. 5). In similar dogs rendered thrombocytopenic with anti-platelet serum, lung ultrastructure was similar to that of controls, although PML's were more frequently seen in capillaries in the former (Fig. 6).

Mitochondria microcrystals deposition in some inherited diseases of lipid metabolism.

1978 ◽  
Vol 36 (2) ◽  
pp. 256-257
Author(s):  
S. Laoussadi ◽  
A. Kahan ◽  
G. Aubouy ◽  
F. Delbarre
Keyword(s):  
Synovial Tissue ◽  
Storage Disease ◽  
Metabolic Diseases ◽  
Uranyl Acetate ◽  
List Type ◽  
Type Ii ◽  
Type Number ◽  
Lipid Storage Disease ◽  
Thin Sections ◽  
Mean Size

Several patients with Fabry's, Gaucher's diseases and hyperlipoproteinemia type II and with arthropatic manifestations were observed.As no histological explanation for these symptoms was available,an ultrastructural study of synovial tissue was done to establish an anatomoclinical relation.Material and Methods :synovial membrane samples were obtained by needle biopsies of the knee from three patients with arthropatic manifestations of each disease.They were fixed in 5% glutaraldehyde, postfixed in 1% osmium tetraoxyde and embedded in Epon 812. Thin sections coloured by uranyl acetate and lead citrate were observed with an Elmiskop I Siemens electron microscope.Two important phenomena were observed in synovial tissue:Specific patterns of each lipid storage disease,which are now well known.In all the three metabolic diseases, hydroxyapatite-like crystals were found. They are characterized by their intramitochondrial localization, without any relation with cristae,an anarchic disposition and a mean size of 550 A.Crystals may be found also free in the cytoplasm of synoviocytes Some micrographs suggest an evolution in four steps :a. mitochondria with only a few microcrystalsb. mitochondria stuffed with these structuresc. disruption of mitochondria membranesd. microcrystals appear free in the cytoplasm

Labelling of non-A, non-B hepatitis infected chimpanzee hepatocytes with nuclease-gold conjugates

1984 ◽  
Vol 42 ◽  
pp. 250-251
Author(s):  
G. D. Gagne ◽  
M. F. Miller ◽  
D. A. Peterson
Keyword(s):  
Endoplasmic Reticulum ◽  
Experimental Infection ◽  
Uranyl Acetate ◽  
Type I ◽  
Cellular Injury ◽  
Type Ii ◽  
Tubular Structures ◽  
Type Iii ◽  
Type Iv ◽  
Infected Chimpanzee

Experimental infection of chimpanzees with non-A, non-B hepatitis (NANB) or with delta agent hepatitis results in the appearance of characteristic cytoplasmic alterations in the hepatocytes. These alterations include spongelike inclusions (Type I), attached convoluted membranes (Type II), tubular structures (Type III), and microtubular aggregates (Type IV) (Fig. 1). Type I, II and III structures are, by association, believed to be derived from endoplasmic reticulum and may be morphogenetically related. Type IV structures are generally observed free in the cytoplasm but sometimes in the vicinity of type III structures. It is not known whether these structures are somehow involved in the replication and/or assembly of the putative NANB virus or whether they are simply nonspecific responses to cellular injury. When treated with uranyl acetate, type I, II and III structures stain intensely as if they might contain nucleic acids. If these structures do correspond to intermediates in the replication of a virus, one might expect them to contain DNA or RNA and the present study was undertaken to explore this possibility.

Interfacial studies in Nb3Sn superconductors

1991 ◽  
Vol 49 ◽  
pp. 590-591
Author(s):  
Ernest L. Hall ◽  
Lee E. Rumaner ◽  
Mark G. Benz
Keyword(s):  
Grain Size ◽  
Grain Boundaries ◽  
Flux Pinning ◽  
Size Control ◽  
High Magnetic Fields ◽  
Type Ii ◽  
Reaction Interface ◽  
Liquid Diffusion ◽  
Type Ii Superconductor ◽  
Grain Size Control

The intermetallic compound Nb3Sn is a type-II superconductor of interest because it has high values of critical current density Jc in high magnetic fields. One method of forming this compound involves diffusion of Sn into Nb foil containing small amounts of Zr and O. In order to maintain high values of Jc, it is important to keep the grain size in the Nb3Sn as small as possible, since the grain boundaries act as flux-pinning sites. It has been known for many years that Zr and O were essential to grain size control in this process. In previous work, we have shown that (a) the Sn is transported to the Nb3Sn/Nb interface by liquid diffusion along grain boundaries; (b) the Zr and O form small ZrO2 particles in the Nb3Sn grains; and (c) many very small Nb3Sn grains nucleate from a single Nb grain at the reaction interface. In this paper we report the results of detailed studies of the Nb3Sn/Nb3Sn, Nb3Sn/Nb, and Nb3Sn/ZrO2 interfaces.

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