Guojun Zheng

Characterization of a recombinant (−)γ-lactamase from Microbacterium hydrocarbonoxydans

A (−)γ-lactamase, Mhg, from Microbacterium hydrocarbonoxydans was over-expressed in E. coli and was characterized after purification. The maximum activity was at pH 8.0 and 60°C and the half life of Mhg was ~30xa0min at 75°C. The enzyme was activated by DTT. The catalytic triad of the (−)γ-lactamase is comprised of residues Ser98, Asp230, and His259 and an oxyanion hole was formed by Tyr32 and Met99 according to the alignment results. Under native conditions, the (−)γ-lactamase consists of two 31xa0kDa homodimers.

An intracellular esterase from Bacillus cereus catalyzing hydrolysis of 1-chloro-3-(1-napthyloxy)-2-acetoxypropanol

An esterase from Bacillus cereus that hydrolyzes 1-chloro-3-(1-napthyloxy)-2-acetoxypropanol was purified to homogeneity. After purification, the molecular mass of the esterase was determined as 43xa0kDa by SDS-PAGE, and estimated as 45xa0kDa using gel filtration, suggesting that the enzyme is a monomer. The optimum pH and temperature for activity of the enzyme were 7.0 and 40°C respectively. The N-terminal sequence was determined. Whole cells of this strain were applied to the resolution of 1-chloro-3-(1-napthyloxy)-2-acetoxypropanol.

Purification and characterization of a novel (-) gamma-lactamase from Microbacterium hydrocarbonoxydans

A (−) gamma-lactamase fromMicrobacterium hydrocarbonoxydans was purified to homogeneity by chromatography methods. SDS-PAGE showed the molecular weight of the enzyme was about 31 kDa. The purified enzyme had a specific activity of 61.3±2.5 U mg−1 for 2-azabicyclo [2.2.1] hept-5-en-3-one [(−) gamma-lactam]. The enantioselectivity factor (E) of the purified enzyme was 9.5±0.8 for unreacted (+) gamma-lactam. TheKm andVmax value were 2.3±0.2 mM and 80.0±15.4 U mg−1 respectively. The highest activity was found at 30 °C and pH 8.0. ESIMS mass spectrometry analysis results and N-terminal sequence indicated the (−) gamma-lactamase might be a new enzyme.

Structural insights into the γ-lactamase activity and substrate enantioselectivity of an isochorismatase-like hydrolase from Microbacterium hydrocarbonoxydans

(+)-γ-lactamase catalyzes the specific hydrolysis of (+)-γ-lactam out of the racemic γ-lactam (2-Azabicyclo[2.2.1]hept-5-en-3-one) to leave optically pure (−)-γ-lactam, which is the key building block of antiviral drugs such as carbovir and abacavir. However, no structural data has been reported on how the enzymes bind the γ-lactams and achieve their enantioselectivities. We previously identified an isochorismatase-like hydrolase (IHL, Mh33H4-5540) with (+)-γ-lactamase activity, which constitutes a novel family of γ-lactamase. Here, we first discovered that this enzyme actually hydrolyzed both (+)- and (−)-γ-lactam, but with apparently different specificities. We determined the crystal structures of the apo-form, (+)-γ-lactam bound, and (−)-γ-lactam bound forms of the enzyme. The structures showed that the binding sites of both (+) and (−)-γ-lactam resemble those of IHLs, but the “cover” loop conserved in IHLs is lacking in the enzyme, probably resulting in its incomplete enantioselectivity. Structural, biochemical, and molecular dynamics simulation studies demonstrated that the steric clash caused by the binding-site residues, especially the side-chain of Cys111 would reduce the binding affinity of (−)-γ-lactam and possibly the catalytic efficiency, which might explain the different catalytic specificities of the enantiomers of γ-lactam. Our results would facilitate the directed evolution and application of Mh33H4-5540 in antiviral drug synthesis.

Promiscuous (+)-γ-lactamase activity of an amidase from nitrile hydratase pathway for efficient synthesis of carbocyclic nucleosides intermediate

Based on bioinformatics analysis, the promiscuous (+)-γ-lactamase activity of an amidase was identified in Rhodococcus erythropolis PR4 and found to be involved in the nitrile hydratase pathway. The amidase is highly enantioselective and can be used in the kinetic resolution of the Vince lactam. The known structure provides a rare insight into the catalytic mechanism of (+)-γ-lactamase with absolute chiral selectivity. This lactamase was cloned, purified, biochemically characterized, and demonstrated to be an ideal catalyst for the preparation of carbocyclic nucleosides of pharmaceutical interest. The chiral selectivity of this enzyme was investigated by molecular docking and site-specific mutagenesis, which provides a foundation for further engineering of these versatile biocatalysts.

Construction of an organelle-like nanodevice via supramolecular self-assembly for robust biocatalysts

BackgroundWhen using the microbial cell factories for green manufacturing, several important issues need to be addressed such as how to maintain the stability of biocatalysts used in the bioprocess and how to improve the synthetic efficiency of the biological system. One strategy widely used during natural evolution is the creation of organelles which can be used for regional control. This kind of compartmentalization strategy has inspired the design of artificial organelle-like nanodevice for synthetic biology and “green chemistry”.ResultsMimicking the natural concept of functional compartments, here we show that the engineered thermostable ketohydroxyglutarate aldolase from Thermotoga maritima could be developed as a general platform for nanoreactor design via supramolecular self-assembly. An industrial biocatalyst-(+)-γ-lactamase was selected as a model catalyst and successful encapsulated in the nanoreactor with high copies. These nanomaterials could easily be synthesized by Escherichia coli by heterologous expression and subsequently self-assembles into the target organelle-like nanoreactors both in vivo and in vitro. By probing their structural characteristics via transmission electronic microscopy and their catalytic activity under diverse conditions, we proved that these nanoreactors could confer a significant benefit to the cargo proteins. The encapsulated protein exhibits significantly improved stability under conditions such as in the presence of organic solvent or proteases, and shows better substrate tolerance than free enzyme.ConclusionsOur biodesign strategy provides new methods to develop new catalytically active protein-nanoreactors and could easily be applied into other biocatalysts. These artificial organelles could have widely application in sustainable catalysis, synthetic biology and could significantly improve the performance of microbial cell factories.Graphical Abstract

Engineering the Enantioselectivity and Thermostability of a (+)-γ-Lactamase from Microbacterium hydrocarbonoxydans for Kinetic Resolution of Vince Lactam (2-Azabicyclo[2.2.1]hept-5-en-3-one)

ABSTRACT To produce promising biocatalysts, natural enzymes often need to be engineered to increase their catalytic performance. In this study, the enantioselectivity and thermostability of a (+)-γ-lactamase from Microbacterium hydrocarbonoxydans as the catalyst in the kinetic resolution of Vince lactam (2-azabicyclo[2.2.1]hept-5-en-3-one) were improved. Enantiomerically pure (−)-Vince lactam is the key synthon in the synthesis of antiviral drugs, such as carbovir and abacavir, which are used to fight against HIV and hepatitis B virus. The work was initialized by using the combinatorial active-site saturation test strategy to engineer the enantioselectivity of the enzyme. The approach resulted in two mutants, Val54Ser and Val54Leu, which catalyzed the hydrolysis of Vince lactam to give (−)-Vince lactam, with 99.2% (enantiomeric ratio [E] > 200) enantiomeric excess (ee) and 99.5% ee (E > 200), respectively. To improve the thermostability of the enzyme, 11 residues with high temperature factors (B-factors) calculated by B-FITTER or high root mean square fluctuation (RMSF) values from the molecular dynamics simulation were selected. Six mutants with increased thermostability were obtained. Finally, the mutants generated with improved enantioselectivity and mutants evolved for enhanced thermostability were combined. Several variants showing (+)-selectivity (E value > 200) and improved thermostability were observed. These engineered enzymes are good candidates to serve as enantioselective catalysts for the preparation of enantiomerically pure Vince lactam. IMPORTANCE Enzymatic kinetic resolution of the racemic Vince lactam using (+)-γ-lactamase is the most often utilized means of resolving the enantiomers for the preparation of carbocyclic nucleoside compounds. The efficiency of the native enzymes could be improved by using protein engineering methods, such as directed evolution and rational design. In our study, two properties (enantioselectivity and thermostability) of a γ-lactamase identified from Microbacterium hydrocarbonoxydans were tackled using a semirational design. The protein engineering was initialized by combinatorial active-site saturation test to improve the enantioselectivity. At the same time, two strategies were applied to identify mutation candidates to enhance the thermostability based on calculations from both a static (B-FITTER based on the crystal structure) and a dynamic (root mean square fluctuation [RMSF] values based on molecular dynamics simulations) way. After combining the mutants, we successfully obtained the final mutants showing better properties in both properties. The engineered (+)-lactamase could be a candidate for the preparation of (−)-Vince lactam.