RESUMO
Serine palmitoyltransferase (SPT) catalyzes the pyridoxal-5'-phosphate (PLP)-dependent decarboxylative condensation of l-serine and palmitoyl-CoA to form 3-ketodihydrosphingosine (KDS). Although SPT was shown to synthesize corresponding products from amino acids other than l-serine, it is still arguable whether SPT catalyzes the reaction with d-serine, which is a question of biological importance. Using high substrate and enzyme concentrations, KDS was detected after the incubation of SPT from Sphingobacterium multivorum with d-serine and palmitoyl-CoA. Furthermore, the KDS comprised equal amounts of 2S and 2R isomers. 1H-NMR study showed a slow hydrogen-deuterium exchange at Cα of serine mediated by SPT. We further confirmed that SPT catalyzed the racemization of serine. The rate of the KDS formation from d-serine was comparable to those for the α-hydrogen exchange and the racemization reaction. The structure of the d-serine-soaked crystal (1.65 Å resolution) showed a distinct electron density of the PLP-l-serine aldimine, interpreted as the racemized product trapped in the active site. The structure of the α-methyl-d-serine-soaked crystal (1.70 Å resolution) showed the PLP-α-methyl-d-serine aldimine, mimicking the d-serine-SPT complex prior to racemization. Based on these enzymological and structural analyses, the synthesis of KDS from d-serine was explained as the result of the slow racemization to l-serine, followed by the reaction with palmitoyl-CoA, and SPT would not catalyze the direct condensation between d-serine and palmitoyl-CoA. It was also shown that the S. multivorum SPT catalyzed the racemization of the product KDS, which would explain the presence of (2R)-KDS in the reaction products.
Assuntos
Serina C-Palmitoiltransferase , Serina , Sphingobacterium , Domínio Catalítico , Cristalização , Medição da Troca de Deutério , Elétrons , Hidrogênio/metabolismo , Palmitoil Coenzima A/metabolismo , Serina/análogos & derivados , Serina/metabolismo , Serina C-Palmitoiltransferase/química , Serina C-Palmitoiltransferase/metabolismo , Sphingobacterium/enzimologia , Sphingobacterium/metabolismo , Esfingosina/análogos & derivados , Esfingosina/biossíntese , Esfingosina/metabolismo , Estereoisomerismo , Especificidade por SubstratoRESUMO
Biodegradation is an efficient and cost-effective approach to remove residual penicillin G sodium (PGNa) from the environment. In this study, the effective PGNa-degrading strain SQW1 (Sphingobacterium sp.) was screened from contaminated soil using enrichment technique. The effects of critical operational parameters on PGNa degradation by strain SQW1 were systematically investigated, and these parameters were optimized by response surface methodology to maximize PGNa degradation. Comparative experiments found the extracellular enzyme to completely degrade PGNa within 60 min. Combined with whole genome sequencing of strain SQW1 and LC-MS analysis of degradation products, penicillin acylase and ß-lactamase were identified as critical enzymes for PGNa biodegradation. Moreover, three degradation pathways were postulated, including ß-lactam hydrolysis, penicillin acylase hydrolysis, decarboxylation, desulfurization, demethylation, oxidative dehydrogenation, hydroxyl reduction, and demethylation reactions. The toxicity of PGNa biodegradation intermediates was assessed using paper diffusion method, ECOSAR, and TEST software, which showed that the biodegradation products had low toxicity. This study is the first to describe PGNa-degrading bacteria and detailed degradation mechanisms, which will provide new insights into the PGNa biodegradation.
Assuntos
Penicilina Amidase , Sphingobacterium , Sphingobacterium/genética , Sphingobacterium/metabolismo , Penicilina Amidase/metabolismo , Penicilina G , Biodegradação AmbientalRESUMO
Environmental challenges arising from organic pollutants pose a significant problem for modern societies. Efficient microbial resources for the degradation of these pollutants are highly valuable. In this study, the bacterial community structure of sludge samples from Taozi Lake (polluted by urban sewage) was studied using 16S rRNA sequencing. The bacterial phyla Proteobacteria, Bacteroidetes, and Chloroflexi, which are potentially important in organic matter degradation by previous studies, were identified as the predominant phyla in our samples, with relative abundances of 48.5%, 8.3%, and 6.6%, respectively. Additionally, the FAPROTAX and co-occurrence network analysis suggested that the core microbial populations in the samples may be closely associated with organic matter metabolism. Subsequently, sludge samples from Taozi Lake were subjected to enrichment cultivation to isolate organic pollutant-degrading microorganisms. The strain Sphingobacterium sp. GEMB-CSS-01, tolerant to sulfanilamide, was successfully isolated. Subsequent investigations demonstrated that Sphingobacterium sp. GEMB-CSS-01 efficiently degraded the endocrine-disrupting compound 17ß-Estradiol (E2). It achieved degradation efficiencies of 80.0% and 53.5% for E2 concentrations of 10 mg/L and 20 mg/L, respectively, within 10 days. Notably, despite a reduction in degradation efficiency, Sphingobacterium sp. GEMB-CSS-01 retained its ability to degrade E2 even in the presence of sulfanilamide concentrations ranging from 50 to 200 mg/L. The findings of this research identify potential microbial resources for environmental bioremediation, and concurrently provide valuable information about the microbial community structure and patterns within Taozi Lake.
Assuntos
Poluentes Ambientais , Sphingobacterium , Esgotos/microbiologia , Sphingobacterium/genética , Sphingobacterium/metabolismo , Lagos/microbiologia , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Estradiol/metabolismo , Biodegradação Ambiental , Poluentes Ambientais/metabolismo , SulfanilamidasRESUMO
Polyphosphate kinase plays an important role in the catalytic synthesis of ATP in vitro. In order to find a polyphosphate kinase that can efficiently synthesize ATP using short-chain polyphosphate (polyP) as substrate, the polyphosphate kinase 2 (PPK2) from Sphingobacterium siyangensis was cloned and expressed in Escherichia coli BL21(DE3). As an enzyme for ATP regeneration, PPK2 was used in combination with l-amino acid ligase (YwfE) to produce l-alanyl-l-glutamine (Ala-Gln). The length of ppk2 of S. siyangensis is 810 bp, encoding 270 amino acids. The SDS-PAGE showed that PPK2 was expressed correctly and its molecular weight was 29.7 kDa as expected. The reaction conditions of PPK2 were optimized. PPK2 could maintain good activity in the range of 22-42 ℃ and pH 7-10. The highest enzyme activity was observed at 37 ℃, pH 7, 30 mmol/L magnesium ion (Mg2+), 5 mmol/L ADP and 10 mmol/L sodium hexametaphosphate, and the yield of ATP reached 60% of the theoretical value in 0.5 hours at this condition. When used in combination with YwfE to produce Ala-Gln, the PPK2 showed a good applicability as an ATP regeneration system, and the effect was similar to that of direct addition of ATP. The PPK2 from S. siyangensis shows good performance in a wide range of temperature and pH, synthesizes ATP with cheap and readily available short chain polyP as substrate. The PPK2 thus provides a new enzyme source for ATP dependent catalytic reaction system.