Propeptide in Rhizopus chinensis Lipase: New Insights into Its Mechanism of Activity and Substrate Selectivity by Computational Design

Abstract

Most fungal lipases contain a propeptide, which is very important for their function and substrate selectivity. In the present study, Rhizopus chinensis lipase (RCL) was used as a research model to explore the mechanism of the propeptide of the lipase. Conventional molecular dynamics (MD) and metadynamics simulations were used to explore the mechanism by which the propeptide affects the activity of the lipase, which was subsequently verified by mutation experiments. MD simulations indicated that the propeptide had an inhibitory effect on the lid movement of RCL and found a key region (Val5-Thr10) on the propeptide. Subsequently, site-directed mutations were created in this region. The mutations enhanced the lipase catalytic efficiency to 700% and showed the potential for the propeptide to shift the substrate specificity of RCL. The specificity and activity of RCL mutants also had similar trends to wild-type RCL toward triglycerides with varying chain lengths. The mutual corroboration of simulation and sitedirected mutagenesis results revealed the vital role of the key propeptide region in the catalytic activity and substrate specificity of the lipase.

MDM-TASK-web: MD-TASK and MODE-TASK web server for analyzing protein dynamics

“…The DCC algorithm is unchanged from that of MD-TASK, with the exception that it is now faster and supports additional atom types. Some of the recent examples from the published literature that used the MD-TASK DCC functionality include [16] , [17] , [18] , [21] .…”

“…Both MD-TASK and MODE-TASK have been highly utilized to mine protein dynamics by offering a series of novel approaches that have demonstrated their applicability in a growing number of cases [6] , [13] , [14] , [15] , [16] , [17] , [18] , [19] , [20] , [21] , [22] , [23] , [24] , [25] , [26] , [27] , [28] , [29] , [30] . Although both software suites are relatively easy to use, the required technical knowledge and software dependencies may act as a hurdle against more widespread usage of the tools and techniques, due to the diversity of operating systems and the relatively fast-evolving Python libraries.…”

Rebuilding the lid region from conformational and dynamic features to engineering applications of lipase in foods: Current status and future prospects

“…Also, Lip2 and BCL exhibited a more complex lid motion, which was described as a "double-domain" motion (Barbe et al, 2009;Cheng, Angkawidjaja, Koga, & Kanaya, 2012;Jiang et al, 2014). In contrast, another recent study has revealed an inhibitory effect of the peptide fragment on lid motion (Wang, Xu, et al, 2021). The above complex structural transitions highlighted the importance of other domains in lid dynamics and lipase catalysis.…”

“…Recently, MD simulation has provided an atomic-level description of lid motion on temporal and energetic scales, thereby predicating the correlation between the lid dynamics/conformation and the function of the lipase. Moreover, molecular simulations allow us to confirm the inhibitory or promoting effects of other regions on the lid motion (Riccardi et al, 2017;Wang, Xu, et al, 2021). By integrating extensive computational simulations and free-energy calculations with mutagenesis and kinetic experiments, a lid-domain-mediated mechanism for substrate selection and binding in lipase catalysis has been elucidated (Xu et al, 2010).…”

Micro-Aqueous Organic System: A Neglected Model in Computational Lipase Design?

“…For the RCL systems, the major differences of RMSF existed in the N-terminal propeptide region. Following a previous study, the N-terminal propeptide of RCL plays an important role in its function [39]. Moreover, the lid region of RCL in the The quantitative root-mean-square fluctuation (RMSF) was evaluated for the backbone atoms of lipases for all systems (Figure 2).…”