Just an additional hydrogen bond can dramatically reduce the catalytic activity of Bacillus subtilis lipase A I12T mutant: An integration of computational modeling and experimental analysis

https://doi.org/10.1016/j.compbiomed.2013.08.018Get rights and content

Abstract

Understanding the structural basis and energetic property of hydrogen bonding and its effects on enzymatic activity is fundamentally important for the rational design of specific enzymes with desired biological functions. In the current study, site-directed mutagenesis analysis preliminarily revealed that the amino acid substitution of Ile12 with Thr12 (I12T) dramatically reduced the hydrolytic activity of Bacillus subtilis lipase A. A further computational investigation proposed that the I12T mutation would establish a geometrically perfect hydrogen bond between the mutated Thr12 and catalytic Ser77 of lipase A, which considerably impaired the catalytic capability of lipase A through two distinct but complementary approaches: rigidizing the enzyme active site and lowering the nucleophilic ability of the catalytic residue Ser77. To verify this hypothesis, a homogenous mutation I12S serving as the control to the I12T mutation was created to examine the hydrogen bonding effect on enzymatic activity. It was found that the I12S mutant only suffered from a slight damage in its hydrolytic ability due to absence of the hydrogen bond originally present at the Thr12–Ser77 interface in the I12T mutant, which was further characterized systematically by quantum mechanics/molecular mechanics (QM/MM) modeling, atom-in-molecules (AIM) analysis and molecular dynamics (MD) simulation. It is suggested that the hydrogen bond arising from the I12T mutation in lipase A can considerably reduce the flexibility and mobility of the enzyme active site, thus impairing the catalytic activity of the lipase A I12T mutant remarkably; the activity loss can be, however, largely recovered by replacing Thr residue at the 12th position of I12T mutant with its analog Ser, which is chemically similar to Thr but cannot form effective hydrogen bonding with Ser77.

Introduction

Enzymes are biological catalysts that allow organisms to carry out biological reactions on timescales compatible with life, speeding up chemical reactions by a factor of 106 to 1020, which represents an amazing enhancement of chemical kinetics with respect to any synthetic catalyst [1]. An understanding of how enzymes work is essential for investigating the biological role of naturally existing enzymes and designing new enzymes [2], [3]. During the process of enzyme catalysis, the associated nonbonded interactions such as hydrogen bonds, Van der Waals contacts and hydrophobic forces play a crucial role in properly locating the substrate within the enzymatic active pocket, stabilizing the transition state of enzyme–substrate complexes, and helping to release products from the catalytic reaction [4]. Among these diverse weak interactions, hydrogen bonding is particularly important because it has been observed to be intensively present around the active site of various enzymes and functions as a director to specify the catalytic reaction [5]. Therefore, elucidating the atomic details and energetic properties of hydrogen bonds in enzymatic catalysis has long been an attractive topic in the enzyme engineering community. Recently, Simon et al. have systematically examined the crystal structures of a wide spectrum of enzyme–substrate complexes, they concluded that the hydrogen bonds stabilizing oxygen-centered negative charges within active oxyanion holes are essential for hydrolases to carry out catalytic function [6]. Kim et al. further pointed out that the short strong hydrogen bonds are responsible for specific enzyme–substrate recognition by promoting the partial proton shuttles and charge redistributions of transition structures [2]. Over the past decades, both experimental and theoretical approaches have been intensively applied to explore the structural basis and energetic behavior of hydrogen bonds in enzyme–substrate interactions, in order to gain a clear picture about the molecular mechanism and biological implications underlying hydrogen bond-participating enzyme catalysis processes [7], [8], [9], [10], [11], [12], [13].

In the present work, we combined in silico modeling and in vitro assays to report the adverse impact of an single hydrogen bond on the hydrolytic activity of Bacillus subtilis lipase A I12T mutant. In the procedure, we initially observed a considerable loss in enzymatic activity upon the substitution of lipase A residue Ile12 with Thr. To elucidate the underlying mechanism of this unanticipated phenomenon, a number of sophisticated theoretical approaches, including molecular dynamics (MD) simulation, hybrid quantum mechanics/molecular mechanics (QM/MM) calculation and atoms-in-molecules (AIM) analysis, were employed to model and dissect the structural and energetic details involved in the lipase A I12T mutation. Based upon computational findings a homogenous mutation I12S serving as the reference of I12T was also experimentally created to test the hydrogen bonding hypothesis proposed by theoretical analyses. This work would help to establish hydrogen bond as a potential and effective factor in the context of rational enzyme design.

Section snippets

Experimental details

The B. subtilis lipase A gene was inserted between XhoI and BamHI restriction sites of pET-28a plasmid (Novagen) with an N-terminal (His)6 fusion tag. This recombinant plasmid was used as a template in site-directed mutagenesis by the QuikChange II Site-Directed Mutagenesis Kit (Agilent tecnhnologies, CA). Primer design and thermal cycling were performed as per the manufacturer's protocol. Briefly, primers having appropriate melting temperature were designed for the region flanking the target

Results and discussion

First, the full length (546 bp) of B. subtilis lipase A gene was successfully amplified from the B. subtilis genome with PCR. The resultant sequence was cloned into the pET-28a vector and then confirmed by sequencing. Site-directed mutagenesis was then carried out by using the recombinant plasmid pET-28a-lipase A as template. DNA sequencing verified that the intended mutations had been introduced into each mutant gene. Both the wild type and mutants of lipase A were overexpressed and purified

Conclusions

A combination of multiple methodologies to study the biological function and activity of biomolecules such as proteins, nucleic acids and peptides has long been the central topic in the chemical, biological and medical communities [37], [38], [39], [40], [41], [42], [43]. In this study, the hydrolysis-deficient I12T mutant of B. subtilis lipase A was created via site-directed mutagenesis, which was investigated systematically by a number of computational methods to elucidate the molecular

Conflict of interest statement

There was no conflict of interest.

Acknowledgments

This work was supported by the National Natural Science Funds for Young Scholar (No. 31200602), the China Postdoctoral Science Foundation (No. 2012M521001), the Jiangsu University for Advanced Professionals (No. 1281330021), and the National Basic Research Program of China (973 Program) (Nos. 2011CBA00801 and 2009CB724700).

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