Application of engineered myoglobins for biosynthesis of clofazimine by integration with chemical synthesis.

Significant efforts have been made in the design of artificial metalloenzymes. Myoglobin (Mb), an O2 carrier, has been engineered to exhibit different functions. Herein, we applied a series of engineered Mb mutants with peroxidase activity for biosynthesis of clofazimine (CFZ), a potential drug with a broad-spectrum antiviral activity, by integration with chemical synthesis. Two of those mutants, F43Y Mb and F43Y/T67R Mb, have been shown to efficiently catalyze the oxidative coupling of 2-N-(4-chlorophenyl) benzene-1,2-diamine (N-4-CPBDA) in the presence of H2O2, with 97% yields. The overall catalytic efficiency (kcat/Km) is 46-fold and 82-fold higher than that of WT Mb, respectively. By further combination of this reaction with chemical synthesis, the production of CFZ was accomplished with an isolated yield of 72%. These results showed that engineered Mbs containing the Tyr-heme cross-link (F43Y Mb and F43Y/T67R Mb) exhibit enhanced activity in the oxidative coupling reaction. This study also indicates that the combination of biocatalysis and chemical synthesis avoids the need for the separation of intermediate products, which offers a convenient approach for the total synthesis of the biological compound CFZ.

O2 Carrier Myoglobin Also Exhibits ß-Lactamase Activity That Is Regulated by the Heme Coordination State.

Heme proteins perform a variety of biological functions and also play significant roles in the field of bio-catalysis. The ß-lactamase activity of heme proteins has rarely been reported. Herein, we found, for the first time, that myoglobin (Mb), an O2 carrier, also exhibits novel ß-lactamase activity by catalyzing the hydrolysis of ampicillin. The catalytic proficiency ((kcat/KM)/kuncat) was determined to be 6.25 × 1010, which is much higher than the proficiency reported for designed metalloenzymes, although it is lower than that of natural ß-lactamases. Moreover, we found that this activity could be regulated by an engineered disulfide bond, such as Cys46-Cys61 in F46C/L61C Mb or by the addition of imidazole to directly coordinate to the heme center. These results indicate that the heme active site is responsible for the ß-lactamase activity of Mb. Therefore, the study suggests the potential of heme proteins acting as ß-lactamases, which broadens the diversity of their catalytic functions.

[Treatment of combined hyperlipidemia patients by jiangzhi tongluo soft capsule combined atorvastatin calcium tablet: a clinical study].

OBJECTIVE: To evaluate the efficacy and safety of using Jiangzhi Tongluo Soft Capsule (JTSC) combined with Atorvastatin Calcium Tablet (ACT) or ACT alone in treatment of combined hyperlipidemia. METHODS: A randomized, double blinded, parallel control, and multi-center clinical research design was adopted. Totally 138 combined hyperlipidemia patients were randomly assigned to the combined treatment group (A) and the atorvastatin treatment group (B) by random digit table, 69 in each group. All patients took ACT 20 mg per day. Patients in the A group took JTSC 100 mg each time, 3 times per day. Those in the B group took JTSC simulated agent, 100 mg each time, 3 times per day. The treatment period for all was 8 weeks. Serum levels of triglyceride (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), and high density lipoprotein cholesterol (HDL-C) were observed before treatment, at week 4 and 8 after treatment; and safety was assessed as well. RESULTS: At week 4 and 8 after treatment serum TG decreased by 26.69% and 33.29% respectively in the A group (both P < 0.01), while it was decreased by 25.7% and 22.98% respectively in the B group (both P < 0.01). At week 8 decreased serum TG was obviously higher in the A group than in the B group (P < 0.05). Compared with before treatment, serum levels of LDL-C and TC levels decreased significantly in the two groups (all P < 0.01). There was no statistical difference in the drop-out value and the drop-out rate of serum LDL-C and TC levels (P > 0.05). At week 8 the serum HDL-C level showed an increasing tendency in the two groups. No obvious increase in peptase or creatase occurred in the two groups after treatment. CONCLUSION: JTSC combined with ACT could lower the serum TG level of combined hyperlipidemia patients with safety.