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Effects of altering the properties of an active site in an enzymatic homogeneous catalyst have been extensively reported. However, the possibility of increasing the number of such sites, as commonly done in heterogeneous catalytic materials, remains unexplored, particularly because those have to accommodate appropriate residues in specific configurations. This possibility was investigated by using a serine ester hydrolase as the target enzyme. By using the Protein Energy Landscape Exploration software, which maps ligand diffusion and binding, we found a potential binding pocket capable of holding an extra catalytic triad and oxyanion hole contacts. By introducing two mutations, this binding pocket became a catalytic site. Its substrate specificity, substrate preference, and catalytic activity were different from those of the native site of the wild type ester hydrolase and other hydrolases, due to the differences in the active site architecture. Converting the binding pocket into an extra catalytic active site was proven to be a successful approach to create a serine ester hydrolase with two functional reactive groups. Our results illustrate the accuracy and predictive nature of modern modeling techniques, opening novel catalytic opportunities coming from the presence of different catalytic environments in single enzymes.

Lipases have valuable potential for industrial use, particularly those mostly active against water-insoluble substrates, such as triglycerides composed of long-carbon chain fatty acids. However, in most cases, engineered variants often need to be constructed to achieve optimal performance for such substrates. Protein engineering techniques have been reported as strategies for improving lipase characteristics by introducing specific mutations in the cap domain of esterases or in the lid domain of lipases or through lid domain swapping. Here, we improved the lipase activity of a lipase (WP_075743487.1, or LipMRD) retrieved from the Marine Metagenomics MarRef Database and assigned to the Actinoalloteichus genus. The improvement was achieved through site-directed mutagenesis and by substituting its lid domain (FRGTEITQIKDWLTDA) with that of Rhizopus delemar lipase (previously R. oryzae; UniProt accession number, I1BGQ3) (FRGTNSFRSAITDIVF). The results demonstrated that the redesigned mutants gain activity against bulkier triglycerides, such as glyceryl tridecanoate and tridodecanoate, olive oil, coconut oil, and palm oil. Residue W89 (LipMRD numbering) appears to be key to the increase in lipase activity, an increase that was also achieved with lid swapping. This study reinforces the importance of the lid domains and their amino acid compositions in determining the substrate specificity of lipases, but the generalization of the lid domain swapping between lipases or the introduction of specific mutations in the lid domain to improve lipase activity may require further investigation. [ABSTRACT FROM AUTHOR]

Proteases are abundant in prokaryotic genomes (~10 per genome), but their recovery encounters expression problems, as only 1% can be produced at high levels; this value differs from that of similarly abundant esterases (1–15 per genome), 50% of which can be expressed at good levels. Here, we design a catalytically efficient artificial protease that can be easily produced. The PluriZyme EH1AB1 with two active sites supporting the esterase activity was employed. A Leu24Cys mutation in EH1AB1, remodelled one of the esterase sites into a proteolytic one through the incorporation of a catalytic dyad (Cys24 and His214). The resulting artificial enzyme, EH1AB1C, efficiently hydrolysed (azo)casein at pH 6.5–8.0 and 60–70 °C. The presence of both esterase and protease activities in the same scaffold allowed the one-pot cascade synthesis (55.0 ± 0.6% conversion, 24 h) of L-histidine methyl ester from the dipeptide L-carnosine in the presence of methanol. This study demonstrates that active sites supporting proteolytic activity can be artificially introduced into an esterase scaffold to design easy-to-produce in-one protease-esterase PluriZymes for cascade reactions, namely, the synthesis of amino acid esters from dipeptides. It is also possible to design artificial proteases with good production yields, in contrast to natural proteases that are difficult to express. [ABSTRACT FROM AUTHOR]