Identification and regulation of the catalytic promiscuity of (−)-γ-lactamase from Microbacterium hydrocarbonoxydans

Abstract

Mhg, a previously reported (-)-γ-lactamase from Microbacterium hydrocarbonoxydans, was identified to have perhydrolase activity by combining structure similarity search with activity assays. Kinetic studies illustrated that perhydrolysis was the native activity owing to lower K m and higher k cat/K m values. Experimental evidence showed that both hydrolysis and perhydrolysis reactions took place at the same active center. Engineering of the putative substrate-binding pocket revealed that Leu233 site played a vital role in the aspects of selective catalysis, soluble protein expression level and optimum temperature shift, etc. The mutants L233A, L233P, and L233T retained (-)-γ-lactamase activity but lost perhydrolase activity, while L233M only kept perhydrolase activity. Substitutions of Leu233 could dramatically influence the state of expressed protein. Computational analysis explicitly explained the relationships between mutations and γ-lactamase activity changes. Our investigations demonstrated that it was an efficient method to identify the enzyme catalytic promiscuity by combining 3D structure alignment with activity validations, and engineering of substrate-binding pocket could serve as a promising way to regulate activities of promiscuous enzymes.

Identification of Cbp1, a c-di-GMP Binding Chemoreceptor in Azorhizobium caulinodans ORS571 Involved in Chemotaxis and Nodulation of the Host Plant

“…Plasmid pBAD/M with a C-terminal 6×His tag was used to overexpress Cbp1 in E. coli Top10 (Sun et al, 2015). A DNA fragment encoding Cbp1 was amplified from A. caulinodans ORS571 genomic DNA with the primer pair Cbp1- Nde I-F/ Hin dIII-R and cloned into the Nde I/ Hin dIII site of pBAD/M to create the recombinant expression plasmid.…”

“…The Cbp1R320A mutant was generated using the QuikChange ® Site-Directed Mutagenesis Kit (Stratagene) with the primer pair Cbp1R320A-F/R. Protein expression was induced with 0.1% L-arabinose and incubation at 16°C for 24 h. Protein purification was performed as described previously (Sun et al, 2015). Proteins were analyzed by SDS-PAGE and their concentration was measured using the standard BCA protein assay.…”

Facilitating the Evolution of Esterase Activity from a Promiscuous Enzyme (Mhg) with Catalytic Functions of Amide Hydrolysis and Carboxylic Acid Perhydrolysis by Engineering the Substrate Entrance Tunnel

“…1) and that its catalytic efficiency as a perhydrolase was 10-fold higher than as a ␥-lactamase. Active site mutagenesis confirmed that the two reactions occur in the same active center (15). Structure analysis revealed that Mhg shares high structural similarity with an aryl esterase from Pseudomonas fluorescens and that both have the same Ser-His-Asp catalytic triad.…”

Biocatalytic routes to anti-viral agents and their synthetic intermediates

“…Over the past 30 years, numerous lactamases have been isolated and applied in the ring-opening of Vince-lactam. [103][104][105][106][107][108][109][110][111][112][113][114][115][116][117][118] Resolution could, alternatively, be achieved using a variety of other enzymes including CAL-B and a non-haem chloroperoxidase from Streptomyces viridochromogenes. [119][120][121][122] CAL-B resolution efficiency could be increased by making use of the activated N-hydroxymethyl Vince-lactam.…”