Murine erythroid 5-aminolevulinate synthase: Adenosyl-binding site Lys221 modulates substrate binding and catalysis

Abstract5-Aminolevulinate synthase (ALAS) catalyzes the initial step of mammalian heme biosynthesis, the condensation between glycine and succinyl-CoA to produce CoA, CO2, and 5-aminolevulinate. The crystal structure of Rhodobacter capsulatus ALAS indicates that the adenosyl moiety of succinyl-CoA is positioned in a mainly hydrophobic pocket, where the ribose group forms a putative hydrogen bond with Lys156. Loss-of-function mutations in the analogous lysine of human erythroid ALAS (ALAS2) cause X-linked sideroblastic anemia. To characterize the contribution of this residue toward catalysis, the equivalent lysine in murine ALAS2 was substituted with valine, eliminating the possibility of a hydrogen bond. The K221V substitution produced a 23-fold increase in the KmSCoA and a 97% decrease in kcat/KmSCoA. This reduction in the specificity constant does not stem from lower affinity toward succinyl-CoA, since the KdSCoA of K221V is lower than that of wild-type ALAS. For both enzymes, the KdSCoA value is significantly different from the KmSCoA. That K221V has stronger binding affinity for succinyl-CoA was further deduced from substrate protection studies, as K221V achieved maximal protection at lower succinyl-CoA concentration than wild-type ALAS. Moreover, it is the CoA, rather than the succinyl moiety, that facilitates binding of succinyl-CoA to wild-type ALAS, as evident from identical KdSCoA and KdCoA values. Transient kinetic analyses of the K221V-catalyzed reaction revealed that the mutation reduced the rates of quinonoid intermediate II formation and decay. Altogether, the results imply that the adenosyl-binding site Lys221 contributes to binding and orientation of succinyl-CoA for effective catalysis

Facile and selective N-alkylation of gentamicin antibiotics via chemoenzymatic synthesis

The rise and spread of antimicrobial resistance has necessitated the development of novel antimicrobials which are effective against drug resistant pathogens. Aminoglycoside antibiotics (AGAs) remain one of our most effective classes of bactericidal drugs. However, they are challenging molecules to selectively modify by chemical synthesis, requiring the use of extensive protection and deprotection steps leading to long, atom- and step-inefficient synthetic routes. Biocatalytic and chemoenzymatic approaches for the generation of AGA derivatives are of interest as they allow access to more concise and sustainable synthetic routes to novel compounds. This work presents a two-step chemoenzymatic route to regioselectively modify the C-6′ position of AGAs. The approach uses a transaminase enzyme to generate an aldehyde on the C-6′ position in the absence of protecting groups, followed by reductive amination to introduce substituents selectively on this position. Seven candidate transaminases were tested for their ability to deaminate a panel of commercially available AGAs. The C-6′ transaminases could deaminate both pseudo di- and trisaccharide AGAs and tolerate the presence or absence of hydroxyl groups on the C-3′- and C-4′-positions. Additionally, sugar substituents on the C-6 hydroxyl were accepted but not on the C-5 hydroxyl. The most promising enzyme, GenB4, was then coupled with a reductive amination step to synthesise eleven novel 6′-gentamicin C1a analogues with conversions of 13–90%. Five of these compounds were active antimicrobials and four of these retained activity against an aminoglycoside-resistant Escherichia coli. This approach allows facile and step-efficient access to novel aminoglycoside compounds under mild reaction conditions and could potentially enable the development of greener, sustainable, and more cost-effective syntheses of novel AGAs

Fauna of monogenean trematodes-parasites of some cyprinid fishes from lake Prespa, Macedonia

During parasitological investigations six species of monogenean trematodes were found on the gills of three cyprinid fish species from Lake Prespa (Macedonia), as follows: Dactylogyrus prostae and Dactylogyrus sphyrna in Leuciscus cephalus albus, Dactylogyrus erhardovae, Dactylogyrus sphyrna and Paradiplozoon zeller in Rutilus rubilio prespensis and Dactylogyrus elegantis and Dactylogyrus vistulae in Chondrostoma nasus prespensis. The prevalence in Leuciscus cephalus albus was 62.22%, in Rutilus rubilio prespensis 59.87% while in Chondrostoma nasus prespensis it was the lowest and amounted to 41.59%. The overall, prevalence of monogeneans in the investigated cyprinid fishes from Lake Prespa was 53.65%, and the mean intensity of infestation was 6.08. Among the monogenean species the highest prevalence occurred with Dactylogyrus sphyrna (25.08%), and the greatest intensity of infestation was evident in the cases of infestation with Dactylogyrus erhardovae (12.87). The greatest pathological effect was associated with the monogeneans Dactylogyrus vistulae and Paradiplozoon zeller. All monogenean species found represented the first record for such parasite fauna of fishes in Macedonia.U toku parazitoloških istraživanja ciprinidnih riba iz Prespanskog jezera (Makedonija), kod 3 vrste riba na škrgama pronađeno je 6 vrsta monogenih trematoda, i to: kod Leuciscus cephalus albus su nađeni Dactylogyrus prostae i Dactylogyrus sphyrna, kod Rutilus rubilio prespensis su pronađeni Dactylogyrus erhardovae, Dactylogyrus sphyrna i Paradiplozoon zeller, kod Chondrostoma nasus prespensis su pronađeni Dactylogyrus elegantis i Dactylogyrus vistulae. Ekstenzitet infestacije kod Leuciscus cephalus albus iznosio je 62,22%, kod Rutilus rubilio prespensis 59,87%, a kod Chondrostoma nasus prespensis je bio najniži i iznosio je 41,59%. Ukupno, ekstenzitet infestacije sa monogeneama kod ispitivanih ciprinidnih riba Prespanskog jezera iznosio je 53,65%, a intenzitet infestacije 6,08. Pojedinačno, po vrstama monogenea, najviši ekstenzitet infestacije je bio sa Dactylogyrus sphyrna (25,08%), a najviši intenzitet infestacije je utvrđen u slučajevima infestacije sa Dactylogyrus erhardovae (12,87). Najveći patogeni uticaj su imale monogene trematode Dactylogyrus vistulae i Paradiplozoon zeller. Sve pronađene vrste monogenea predstavljaju prvi nalaz za parazitofaunu riba Makedonije.nul

DEFINING THE BREED STANDARDS AND BREEDING GOALS FOR DOMESTIC MOUNTAIN HORSE

Domestic mountain horse is a transpoundary breed present in many Balkan countries. According to the Low of Animal Production of R. of Macedonia from 2008, for every breed of domestic animal in our state is nessesery to create a separate breeding program. In 2013 in low of animal production the autohtonous breeds of equidaes are added two new varietes - domestic mountain horse and domestic donkey. Facing the obtained results from estimating the main morphological, reproductive and functional traits in 220 domestic mountain horses in central mountain part of Macedonia during 2015 and 2016, the current standards and breeding goals were defined for the next national breeding program of this breed. According to the fact that the domestic mountain horse is a metapopulation and transboundary breed of horse in almost all Balkan countries, it is a good recommendation to create a regional breeding program with flexible standards and breeding goals for this breed of horses for all Balkan countries together

Discocotyle ohridana n. Sp. (monogenea: Discocotylidae) on the gills of ohrid belvica salmothymus ohridanus (pisces: Salmonidae) from the lake ohrid, macedonia

Discocotyle ohridana n.sp. is described from the gills of Ohrid belvica Salmothymus ohridanus Steindachner, 1892; (syn. Acantholingua ohridana Hadzisce, 1961) from the Lake Ohrid, Macedonia. According our investigation the new species D. ohridana n.sp. differs 56.47% of observed S. ohridanus have been infected and average intensity of infestation is 2.84

History-based verification of functional behaviour of concurrent programs

Modular verification of the functional behaviour of a concurrent program remains a challenge. We propose a new way to achieve this, using histories, modelled as process algebra terms, to keep track of local changes. When threads terminate or synchronise in some other way, local histories are combined into global histories, and by resolving the global histories, the reachable state properties can be determined. Our logic is an extension of permission-based separation logic, which supports expressive and intuitive specifications. We discuss soundness of the approach, and illustrate it on several examples

Mechanoenzymatic reactions for the hydrolysis of PET

Recent advances in the enzymatic degradation of poly(ethylene terphthalate) (PET) have led to a number of PET hydrolytic enzymes and mutants being developed. With the amount of PET building up in the natural world, there is a pressing need to develop scalable methods of breaking down the polymer into its monomers for recycling or other uses. Mechanoenzymatic reactions have gained traction recently as a green and efficient alternative to traditional biocatalytic reactions. For the first time we report increased yields of PET degradation by whole cell PETase enzymes by up to 27-fold by utilising ball milling cycles of reactive aging, when compared with typical solution-based reactions. This methodology leads to up to a 2600-fold decrease in the solvent required when compared with other leading degradation reactions in the field and a 30-fold decrease in comparison to reported industrial scale PET hydrolysis reactions

Mitochondrial protein import: precursor oxidation in a ternary complex with disulfide carrier and sulfhydryl oxidase

The biogenesis of mitochondrial intermembrane space proteins depends on specific machinery that transfers disulfide bonds to precursor proteins. The machinery shares features with protein relays for disulfide bond formation in the bacterial periplasm and endoplasmic reticulum. A disulfide-generating enzyme/sulfhydryl oxidase oxidizes a disulfide carrier protein, which in turn transfers a disulfide to the substrate protein. Current views suggest that the disulfide carrier alternates between binding to the oxidase and the substrate. We have analyzed the cooperation of the disulfide relay components during import of precursors into mitochondria and identified a ternary complex of all three components. The ternary complex represents a transient and intermediate step in the oxidation of intermembrane space precursors, where the oxidase Erv1 promotes disulfide transfer to the precursor while both oxidase and precursor are associated with the disulfide carrier Mia40

Fauna of monogenean trematodes-parasites of some cyprinid fishes from lake Prespa, Macedonia

During parasitological investigations six species of monogenean trematodes were found on the gills of three cyprinid fish species from Lake Prespa (Macedonia), as follows: Dactylogyrus prostae and Dactylogyrus sphyrna in Leuciscus cephalus albus, Dactylogyrus erhardovae, Dactylogyrus sphyrna and Paradiplozoon zeller in Rutilus rubilio prespensis and Dactylogyrus elegantis and Dactylogyrus vistulae in Chondrostoma nasus prespensis. The prevalence in Leuciscus cephalus albus was 62.22%, in Rutilus rubilio prespensis 59.87% while in Chondrostoma nasus prespensis it was the lowest and amounted to 41.59%. The overall, prevalence of monogeneans in the investigated cyprinid fishes from Lake Prespa was 53.65%, and the mean intensity of infestation was 6.08. Among the monogenean species the highest prevalence occurred with Dactylogyrus sphyrna (25.08%), and the greatest intensity of infestation was evident in the cases of infestation with Dactylogyrus erhardovae (12.87). The greatest pathological effect was associated with the monogeneans Dactylogyrus vistulae and Paradiplozoon zeller. All monogenean species found represented the first record for such parasite fauna of fishes in Macedonia.U toku parazitoloških istraživanja ciprinidnih riba iz Prespanskog jezera (Makedonija), kod 3 vrste riba na škrgama pronađeno je 6 vrsta monogenih trematoda, i to: kod Leuciscus cephalus albus su nađeni Dactylogyrus prostae i Dactylogyrus sphyrna, kod Rutilus rubilio prespensis su pronađeni Dactylogyrus erhardovae, Dactylogyrus sphyrna i Paradiplozoon zeller, kod Chondrostoma nasus prespensis su pronađeni Dactylogyrus elegantis i Dactylogyrus vistulae. Ekstenzitet infestacije kod Leuciscus cephalus albus iznosio je 62,22%, kod Rutilus rubilio prespensis 59,87%, a kod Chondrostoma nasus prespensis je bio najniži i iznosio je 41,59%. Ukupno, ekstenzitet infestacije sa monogeneama kod ispitivanih ciprinidnih riba Prespanskog jezera iznosio je 53,65%, a intenzitet infestacije 6,08. Pojedinačno, po vrstama monogenea, najviši ekstenzitet infestacije je bio sa Dactylogyrus sphyrna (25,08%), a najviši intenzitet infestacije je utvrđen u slučajevima infestacije sa Dactylogyrus erhardovae (12,87). Najveći patogeni uticaj su imale monogene trematode Dactylogyrus vistulae i Paradiplozoon zeller. Sve pronađene vrste monogenea predstavljaju prvi nalaz za parazitofaunu riba Makedonije.nul