Unnatural activities and mechanistic insights of cytochrome P450 PikC gained from site-specific mutagenesis by non-canonical amino acids.

Cytochrome P450 enzymes play important roles in the biosynthesis of macrolide antibiotics by mediating a vast variety of regio- and stereoselective oxidative modifications, thus improving their chemical diversity, biological activities, and pharmaceutical properties. Tremendous efforts have been made on engineering the reactivity and selectivity of these useful biocatalysts. However, the 20 proteinogenic amino acids cannot always satisfy the requirement of site-directed/random mutagenesis and rational protein design of P450 enzymes. To address this issue, herein, we practice the semi-rational non-canonical amino acid mutagenesis for the pikromycin biosynthetic P450 enzyme PikC, which recognizes its native macrolide substrates with a 12- or 14-membered ring macrolactone linked to a deoxyamino sugar through a unique sugar-anchoring mechanism. Based on a semi-rationally designed substrate binding strategy, non-canonical amino acid mutagenesis at the His238 position enables the unnatural activities of several PikC mutants towards the macrolactone precursors without any sugar appendix. With the aglycone hydroxylating activities, the pikromycin biosynthetic pathway is rewired by the representative mutant PikCH238pAcF carrying a p-acetylphenylalanine residue at the His238 position and a promiscuous glycosyltransferase. Moreover, structural analysis of substrate-free and three different enzyme-substrate complexes of PikCH238pAcF provides significant mechanistic insights into the substrate binding and catalytic selectivity of this paradigm biosynthetic P450 enzyme.

Comparative biochemical characterization of mammalian-derived CYP11A1s with cholesterol side-chain cleavage activities.

Steroid drugs, the second largest class of pharmaceuticals after antibiotics, have shown significant anti-inflammatory, anti-allergic, and endocrine-regulating effects. A group of cytochrome P450 enzymes, namely, CYP11A1 isoenzymes from different organisms are capable of converting cholesterol into pregnenolone, which is a pivotal reaction in both steroid metabolism and (bio)synthetic network of steroid products. However, the low activity of CYP11A1s greatly restricts the industrial application of these cholesterol side-chain cleavage enzymes. Herein, we investigate ten CYP11A1 enzymes of different origins and in vitro characterize two CYP11A1s with a relatively higher expression level from Capra hircus and Sus scrofa, together with the CYP11A1s from Homo sapiens and Bos taurus as references. Towards five selected sterol substrates with different side chain structures, S. scrofa CYP11A1 displays relatively higher activities. Through redox partners combination screening, we reveal the optimal redox partner pair of S. scrofa adrenodoxin and C. hircus adrenodoxin reductase. Moreover, the semi-rational mutagenesis for the active sites and substrate entrance channels of human and bovine CYP11A1s is performed based on comparative analysis of their crystal structures. The mutant mBtCYP11A1-Q377A derived from mature B. taurus CYP11A1 shows a 1.46 times higher activity than the wild type enzyme. These results not only demonstrate the tunability of the highly conserved CYP11A1 isoenzymes, but also lay a foundation for the following engineering efforts on these industrially relevant P450 enzymes.

In vitro analysis of synergistic combination of polymyxin B with 12 other antibiotics against MDR Acinetobacter baumannii isolated from a Chinese tertiary hospital.

In clinical practice, polymyxins are suggested to be used in combination with other antibiotics for improving their antibacterial efficacy and preventing the emergency of antibiotic-resistant strains. However, even though synergistic combination of polymyxin B with many antibiotics have been confirmed in various studies with different bacterial species and analyzing methods, which antibiotic is the best option for combination therapy of polymyxin B against MDR A. baumannii remains uncertain. In this study, we systematically analyzed the synergistic combination of polymyxin B with 12 other antibiotics against MDR A. baumannii isolated from a Chinese tertiary hospital using the checkerboard assay. The results suggest that, for polymyxin B-based combination therapy against MDR A. baumannii as characterized in this hospital, cefperazone-sulbactam may be the best partner, since it has the highest synergistic rate and the best synergistic effect with polymyxin B. Minocycline, imipenem, meropenem, ceftazidime, cefepime, amikacin and sulfamethoxazole also have some synergistic effects with polymyxin B, but piperacillin-tazobactam, ciprofloxacin, levofloxacin and tobramycin show no synergism. None of these 12 antibiotics has an antagonistic effect when combined with polymyxin B.

Synergistic antibacterial and anti-biofilm activities of resveratrol and polymyxin B against multidrug-resistant Pseudomonas aeruginosa.

Bacterial infection caused by multidrug-resistant Pseudomonas aeruginosa has become a challenge in clinical practice. Polymyxins are used as the last resort agent for otherwise untreatable Gram-negative bacteria, including multidrug-resistant P.aeruginosa. However, pharmacodynamic (PD) and pharmacokinetic (PK) data on polymyxins suggest that polymyxin monotherapy is unlikely to generate reliably efficacious plasma concentrations. Also, polymyxin resistance has been frequently reported, especially among multidrug-resistant P.aeruginosa, which further limits its clinical use. A strategy for improving the antibacterial activity of polymyxins and preventing the development of polymyxin resistance is to use polymyxins in combination with other agents. In this study, we have demonstrated that resveratrol, a well tolerated compound, has synergistic effects when tested in vitro with polymyxin B on antibacterial and anti-biofilm activities. However, its' systemic use is limited as the required high plasma levels of resveratrol are not achievable. This suggests that it could be a partner for the combination therapy of polymyxin B in the treatment of topical bacterial infection caused by MDR P.aeruginosa.

The Role of Cross-Pathway Control Regulator CpcA in the Growth and Extracellular Enzyme Production of Penicillium oxalicum.

CpcA is a conserved transcriptional activator for the cross-pathway control of amino acid biosynthetic genes in filamentous fungi. Previous studies of this regulator mainly revealed its function under amino acid starvation condition, where amino acid biosynthetic inhibitors were added in the culture. In this study, the biological function of CpcA in Penicillium oxalicum was investigated under different cultivation conditions. Disruption of cpcA led to decreased cell growth either in the presence or absence of histidine biosynthetic inhibitor, and the phenotype could be rescued by the addition of exogenous amino acid sources. In addition, CpcA was required for the rapid production of cellulase when cells were cultured on cellulose. Transcript abundance measurement showed that a set of amino acid biosynthetic genes as well as two major cellulase genes were significantly down-regulated in cpcA deletion mutant relative to wild type. Taken together, the results revealed the biological role of CpcA in supporting normal growth and extracellular enzyme production of P. oxalicum under amino acid non-starvation condition.

The Indispensable Role of Histone Methyltransferase PoDot1 in Extracellular Glycoside Hydrolase Biosynthesis of Penicillium oxalicum.

Histone methylation is associated with transcription regulation, but its role for glycoside hydrolase (GH) biosynthesis is still poorly understood. We identified the histone H3 lysine 79 (H3K79)-specific methyltransferase PoDot1 in Penicillium oxalicum. PoDot1 affects conidiation by regulating the transcription of key regulators (BrlA, FlbC, and StuA) of asexual development and is required in normal hyphae septum and branch formation by regulating the transcription of five septin-encoding genes, namely, aspA, aspB, aspC, aspD, and aspE. Tandem affinity purification/mass spectrometry showed that PoDot1 has no direct interaction with transcription machinery, but it affects the expressions of extracellular GH genes extensively. The expression of genes (amy15A, amy13A, cel7A/cbh1, cel61A, chi18A, cel3A/bgl1, xyn10A, cel7B/eg1, cel5B/eg2, and cel6A/cbh2) that encode the top 10 GHs was remarkably downregulated by Podot1 deletion (ΔPodot1). Consistent with the decrease in gene transcription level, the activities of amylases and cellulases were significantly decreased in ΔPodot1 mutants in agar (solid) and fermentation (liquid) media. The repression of GH gene expressions caused by PoDot1 deletion was not mediated by key transcription factors, such as AmyR, ClrB, CreA, and XlnR, but was accompanied by defects in global demethylated H3K79 (H3K79me2) and trimethylated H3K79 (H3K79me3). The impairment of H3K79me2 on specific GH gene loci was observed due to PoDot1 deletion. The results implies that defects of H3K79 methylation is the key reason of the downregulated transcription level of GH-encoding genes and reveals the indispensable role of PoDot1 in extracellular GH biosynthesis.

The Simplified Carbapenem Inactivation Method (sCIM) for Simple and Accurate Detection of Carbapenemase-Producing Gram-Negative Bacilli.

This study reports the simplified carbapenem inactivation method (sCIM) to detect carbapenemase-producing gram-negative bacilli in a simple and accurate manner. This method is based on the modified carbapenem inactivation method (mCIM) with the improvement of experimental procedures. Instead of incubating the antibiotic disk in the organism culture media, the organism to be tested was smeared directly onto the antibiotic disk in the sCIM. For evaluating the sensitivity and specificity of the method, a total of 196 Enterobacteriaceae, 73 Acinetobacter baumannii, and 158 Pseudomonas aeruginosa isolates were collected. Polymerase chain reaction (PCR) was used to detect the carbapenemase genes. Phenotypic evaluations were performed using both the sCIM and the mCIM. PCR results showed that, of the 196 Enterobacteriaceae strains, 147 expressed the carbapenemase genes blaKPC-2 (58.5%), blaIMP-4 (21.8%), blaIMP-2 (2.0%), blaVIM-1 (6.1%), blaNDM-1 (10.2%), and blaOXA-48 (1.4%). sCIM results had high concordance with PCR results (99.5%) and mCIM results (100%) with the exception of one Klebsiella pneumoniae strain, which had an minimal inhibitory concentration (MIC) for imipenem of 0.25 mg/L. PCR demonstrated that 53 of the 73 A. baumannii isolates expressed the carbapenemase genes blaOXA-23 (98.1%) and blaVIM-2 (1.8%). sCIM and PCR results corresponded but all A. baumannii isolates were carbapenemase negative by the mCIM. PCR demonstrated that 25 of the 158 P. aeruginosa isolates expressed carbapenemase genes blaVIM-1 (52%) , blaVIM-2 (8%) , blaVIM-4 (36%), and blaIMP-4 (4%). sCIM results had high concordance with PCR results (100%) and the mCIM results (99.4%) with the exception of one P. aeruginosa isolate that expressed the blaVIM-4 gene. The sCIM offers specificity and sensitivity comparable to PCR but has the advantage of being more user-friendly. This method is suitable for routine use in most clinical microbiology laboratories for the detection of carbapenemase-producing gram-negative bacilli.

Consolidated bioprocessing for sodium gluconate production from cellulose using Penicillium oxalicum.

The feasibility of consolidated bioprocessing for sodium gluconate production from cellulose was studied. A recombinant strain named z19 was constructed from Penicillium oxalicum wild-type strain 114-2 for simultaneous expression of glucose oxidase and catalase from Aspergillus niger. While keeping a cellulolytic ability similar with that of 114-2, z19 secreted certain amounts of glucose oxidase and catalase. Fed-batch and two-stage temperature control strategy (0-120 h, 30 °C; 120-192 h, 45 °C) was utilized for sodium gluconate production from cellulose (filter paper power), with 13.54 g/L of sodium gluconate obtained at the end of the fermentation. The results provide an alternative route for producing sodium gluconate from cellulose in a one-pot reaction.

Hypermethylation of the PRKCZ Gene in Type 2 Diabetes Mellitus.

Objectives. To study the correlation between the methylation of protein kinase C epsilon zeta (PRKCZ) gene promoters and type 2 diabetes mellitus (T2DM). Methods. The case-control method was implemented in 272 unrelated to one another cases in Shiyan People's Hospital. Of those, 152 were diagnosed as T2DM cases, and the other 120 cases were healthy individuals visiting the hospital for a physical examination. The subjects were first divided into two groups: the T2DM group and the normal control (NC) group. Next, methylated DNA immunoprecipitation chip (MeDIP-chip) was used for detection. Bisulfite sequencing PCR (BSP) and gene sequencing were then performed to detect and analyze the correlation between PRKCZ gene promoter methylation and T2DM. Finally, Western blotting was applied to determine the serum level of PRKCZ. The data were then analyzed with the statistics analyzing software SPSS 17.0. Results. In contrast with cases in NC, T2DM patients showed a high level of methylation, with 7 of 9 CpG sites were shown to be methylated, whereas, in the control group, only one CpG site was found to be methylated. The methylated CpG sites for the two groups showed marked differences (P < 0.01). Additionally, the level of PRKCZ was decreased in T2DM subjects, and the difference between the two groups was statistically significant (P < 0.05). Discussion. This study suggests that the PRKCZ gene is the hypermethylated gene of T2DM and the hypermethylation PRKCZ gene may be involved in the pathogenesis of T2DM.

Endothelial nitric oxide synthase gene polymorphisms associated with susceptibility to high altitude pulmonary edema in Chinese railway construction workers at Qinghai-Tibet over 4 500 meters above sea level / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To examine whether the polymorphisms of endothelial nitric oxide synthase (eNOS) gene are associated with the susceptibility to high altitude pulmonary edema (HAPE) in Chinese railway construction workers at Qinghai-Tibet where the altitude is over 4 500 m above sea level.</p><p><b>METHODS</b>A case-control study was conducted including 149 HAPE patients in the construction workers and 160 healthy controls randomly recruited from their co-workers, matching the patients in ethnicity, age, sex, lifestyle, and working conditions. Three polymorphisms of eNOS gene, T-786C in promoter, 894G/T in exon 7, and 27bp variable number tandem repeat (VNTR) in intron 4, were genotyped using polymerase chain reaction (PCR) and confirmed with DNA sequencing.</p><p><b>RESULTS</b>The frequencies of 894T allele and heterozygous G/T of the 894G/T variant were significantly higher in HAPE patients group than in the control group (P=0.0028 and P=0.0047, respectively). However, the frequencies of the T-786C in promoter and the 27bp VNTR in intron 4 were not significantly different between the two groups. Haplotypic analysis revealed that the frequencies of two haplotypes (H3,T-T-b, b indicates 5 repeats of 27 bp VNTR; H6, C-G-a, a indicates 4 repeats of 27 bp VNTR) were significantly higher in HAPE patients (both Pü0.0001). On the contrary, the frequencies of H1 (T-G-b) and H2 (T-G-a) were lower in HAPE patients than in healthy controls (both Pü0.001).</p><p><b>CONCLUSIONS</b>Two haplotypes (T-T-b and C-G-a) may be strongly associated with susceptibility to HAPE. Compared with the individual alleles of eNOS gene, the interaction of multiple genetic markers within a haplotype may be a major determinant for the susceptibility to HAPE.</p>

Genetic interaction of Hsp70 family genes polymorphisms with high-altitude pulmonary edema among Chinese railway constructors at altitudes exceeding 4000 meters.

BACKGROUND: High-altitude pulmonary edema (HAPE) is thought of as an independent clinical disorder with a constitutional or genetic component in its etiology. We focused on 5 common polymorphisms within HSPA1A (rs1043618 and rs1008438), HSPA1B (rs1061581 and rs539689) and HSPA1L (rs2227956) of Hsp70 family to explore their potential interaction upon susceptibility to HAPE in Chinese. METHODS: A total of 148 HAPE patients and 483 matched controls were recruited during the construction of Qinghai-Tibet railway from 2001 to 2006. Genotyping was performed using PCR-RFLP, PCR-SSCP and PCR-direct-sequencing techniques. Promoter activity was evaluated by luciferase reporter assays. Gene-gene interaction was conducted by MDR v.2.0, and haplotype-diplotype analysis by Haplo.stats v.1.4.0. RESULTS: Significant differences were observed in the genotype (P=0.0136) and allele (P=0.0299) distributions of rs1008438, and in rs1061581 allele distribution (P=0.0421) between HAPE patients and controls. Interaction analysis indicated that 3 polymorphisms (rs1061581, rs1043618 and rs1008438) shared strong synergism with a testing accuracy of 0.792 and cross-validation consistency 10 out of 10 (P=0.001). Haplotypes Hap4 (G-C-A, in order of rs1061581, rs1043618 and rs1008438) and Hap5 (G-G-A) had an 86% reduced risk (P=0.0009) against and Hap7 (A-C-C) had a 2.43-fold increased risk for HAPE. When considered as diplotypes, significance was noted for Dip5 (Hap1-Hap7) (OR=3.39; 95% CI: 1.28-9.17; P=0.0140). Functional assessment supported the involvement of rs1008438 in the pathogenesis of HAPE. CONCLUSION: We demonstrated strong interaction of rs1061581, rs1043618 and rs1008438 polymorphisms within Hsp70 family upon susceptibility to HAPE in Chinese. Moreover, polymorphism rs1008438 might cause the development of HAPE via a change in HSPA1A promoter activity.