Probing the Effect of the Non-Active-Site Mutation Y229W in New Delhi Metallo-β-lactamase-1 by Site-Directed Mutagenesis, Kinetic Studies, and Molecular Dynamics Simulations

Jiao, Chen; Chen, Hui; Shi, Yun; Hu, Feng; Lao, Xingzhen; Gao, Xiangdong; Zheng, Heng; Yao, Wenbing

doi:10.1371/journal.pone.0082080

Cited by 28 publications

(24 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It is observed that in addition to enhancement in the levels of expression and solubilization, the K m increased by 1.7-fold, while k cat increased by about 35-fold, for CefF GOS compared to CefF W (wild type). The increase in K m along with the increase in k cat can be attributed to the active-site enlargement, which causes the enzyme to release the product efficiently, coupled with weak substrate binding leading to high k cat and K m (28). The kinetic parameters K m , k cat , and k cat /K m reported in this study for deacetylcephalosporin C synthase correlate well with the reported values for deacetoxycephalosporin C synthase and its substrate analogue penicillin G (20).…”

Section: Resultssupporting

confidence: 84%

Modified Deacetylcephalosporin C Synthase for the Biotransformation of Semisynthetic Cephalosporins

Balakrishnan

Ganesan

Rajasekaran

et al. 2016

Appl Environ Microbiol

View full text Add to dashboard Cite

Deacetylcephalosporin C synthase (DACS), a 2-oxoglutarate-dependent oxygenase synthesized by Streptomyces clavuligerus, transforms an inert methyl group of deacetoxycephalosporin C (DAOC) into an active hydroxyl group of deacetylcephalosporin C (DAC) during the biosynthesis of cephalosporin. It is a step which is chemically difficult to accomplish, but its development by use of an enzymatic method with DACS can facilitate a cost-effective technology for the manufacture of semisynthetic cephalosporin intermediates such as 7-amino-cephalosporanic acid (7ACA) and hydroxymethyl-7-amino-cephalosporanic acid (HACA) from cephalosporin G. As the native enzyme showed negligible activity toward cephalosporin G, an unnatural and less expensive substrate analogue, directed-evolution strategies such as random, semirational, rational, and computational methods were used for systematic engineering of DACS for improved activity. In comparison to the native enzyme, several variants with improved catalytic efficiency were found. The enzyme was stable for several days and is expressed in soluble form at high levels with significantly higher k cat /K m values. The efficacy and industrial scalability of one of the selected variants, CefF GOS , were demonstrated in a process showing complete bioconversion of 18 g/liter of cephalosporin G into deacetylcephalosporin G (DAG) in about 80 min and showed reproducible results at higher substrate concentrations as well. DAG could be converted completely into HACA in about 30 min by a subsequent reaction, thus facilitating scalability toward commercialization. The experimental findings with several mutants were also used to rationalize the functional conformation deduced from homology modeling, and this led to the disclosure of critical regions involved in the catalysis of DACS. IMPORTANCE7ACA and HACA serve as core intermediates for the manufacture of several semisynthetic cephalosporins. As they are expensive, a cost-effective enzyme technology for the manufacture of these intermediates is required. Deacetylcephalosporin C synthase (DACS) was identified as a candidate enzyme for the development of technology from cephalosporin G in this study. Directedevolution strategies were employed to enhance the catalytic efficiency of deacetylcephalosporin C synthase. One of the selected mutants of deacetylcephalosporin C synthase could convert high concentrations of cephalosporin G into DAG, which subsequently could be converted into HACA completely. As cephalosporin G is inexpensive and readily available, the technology would lead to a substantial reduction in the cost for these intermediates upon commercialization. S emisynthetic cephalosporins, a class of ␤-lactam antibiotics, have shown remarkable effectiveness in the treatment of infectious diseases. Together with penicillins, they comprise nearly 65% of anti-infectives used worldwide. Their high specificity and low toxicity, coupled with the evolvability of newer generations of antibiotics, have led to ␤-lactams being by far the most fr...

show abstract

Section: Resultssupporting

confidence: 84%

Modified Deacetylcephalosporin C Synthase for the Biotransformation of Semisynthetic Cephalosporins

Balakrishnan

Ganesan

Rajasekaran

et al. 2016

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…Our study together with the work of Chen et al (8) emphasizes the need to further address the significance of non-active-site residues in the structure, function, and evolution of NDM-1. Such studies will help us to better understand the mechanism by which NDM-1 hydrolyzes ␤-lactam antibiotics and to develop novel inhibitors directed toward specific amino acid residues.…”

Section: Resultsmentioning

confidence: 68%

“…Similarly, the nuclear magnetic resonance (NMR) structures of NDM-1 and cephalosporins show that Trp-93 was positioned perpendicular to the five-or six-membered ring placed close to the cleaved amide bond and stabilized the complex through hydrophobic interactions (26). In another study, Chen et al also elucidated the significance of non-activesite residue Tyr-229 in the catalytic proficiency of NDM-1 by changing it to Trp (8). They found that the Tyr229Trp mutant had higher k cat and K m values than those of wild-type NDM-1, thus resulting in 1-to 7-fold increases in k cat /K m values against different antibiotics.…”

Section: Resultsmentioning

confidence: 98%

“…The relevance of several active-site residues in the catalytic activity of NDM-1 has been studied in detail (7). However, there are only a few reports on the involvement of non-active-site residues in the functional versatility of NDM-1 (8). It is hypothesized that amino acid residues flanking the active site of ␤-lactamases may also play a crucial role in modulating its catalytic efficiency (9).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Role of Non-Active-Site Residue Trp-93 in the Function and Stability of New Delhi Metallo-β-Lactamase 1

Khan

Rehman

2016

Antimicrob Agents Chemother

View full text Add to dashboard Cite

New Delhi metallo-␤-lactamase-1 (NDM-1) is expressed by various members of Enterobacteriaceae as a defense mechanism to hydrolyze ␤-lactam antibiotics. Despite various studies showing the significance of active-site residues in the catalytic mechanism, there is a paucity of reports addressing the role of non-active-site residues in the structure and function of NDM-1. In this study, we investigated the significance of non-active-site residue Trp-93 in the structure and function of NDM-1. We cloned bla NDM-1 from an Enterobacter cloacae clinical strain (EC-15) and introduced the mutation of Trp-93 to Ala (yielding the Trp93Ala mutant) by PCR-based site-directed mutagenesis. Proteins were expressed and purified to homogeneity by affinity chromatography. The MICs of the Trp93Ala mutant were reduced 4-to 8-fold for ampicillin, cefotaxime, ceftazidime, cefoxitin, imipenem, and meropenem. The poor hydrolytic activity of the Trp93Ala mutant was also reflected by its reduced catalytic efficiency. The overall catalytic efficiency of the Trp93Ala mutant was reduced by 40 to 55% (the K m was reduced, while the k cat was similar to that of wild-type NDM-1 [wtNDM-1]). Heat-induced denaturation showed that the ⌬G D o and T m of Trp93Ala mutant were reduced by 1.8 kcal/mol and 4.8°C, respectively. Far-UV circular dichroism (CD) analysis showed that the ␣-helical content of the Trp93Ala mutant was reduced by 2.9%. The decrease in stability and catalytic efficiency of the Trp93Ala mutant was due to the loss of two hydrogen bonds with Ser-63 and Val-73 and hydrophobic interactions with Leu-65, Val-73, Gln-123, and Asp-124. The study provided insight into the role of non-active-site amino acid residues in the hydrolytic mechanism of NDM-1.

show abstract

“…For example, the high flexibility of the substrate-binding site/pocket is necessary for substrate entrance and product egress, and the increased flexibility can also reduce the activation energy necessary to generate reaction intermediates, thus resulting in a more efficient substrate turnover (J. Chen et al, 2013). On the other hand, the presence of flexibility could also increase the substrate binding affinity (Liu, Tao, Meng, Fu, & Zhang, 2011) The stability of a protein is widely believed to arise from its decreased flexibility, implying an inverse correlation between the stability and flexibility (Kamerzell & Middaugh, 2008;Karshikoff, Nilsson, & Ladenstein, 2015).…”

Section: Discussionmentioning

confidence: 99%

In silico screening, molecular docking, and molecular dynamics studies of SNP-derived human P5CR mutants

Sang

et al. 2016

Journal of Biomolecular Structure and Dynamics

View full text Add to dashboard Cite

Pyrroline-5-carboxylate reductase (P5CR) encoded by PYCR1 gene is a housekeeping enzyme that catalyzes the reduction of P5C to proline using NAD(P)H as the cofactor. In this study, we used in silico approaches to examine the role of nonsynonymous single-nucleotide polymorphisms in the PYCR1 gene and their putative functions in the pathogenesis of Cutis Laxa. Among the 348 identified SNPs, 15 were predicted to be potentially damaging by both SIFT and PolyPhen tools; of them two SNP-derived mutations, R119G and G206W, have been previously reported to correlate with Cutis Laxa. These two mutations were therefore selected to be mapped to the wild-type (WT) P5CR structure for further structural and functional analyses. The results of comparative computational analyses using I-Mutant and Autodock reveal reductions in both stability and cofactor binding affinity of these two mutants. Comparative molecular dynamics (MD) simulations were performed to evaluate the changes in dynamic properties of P5CR upon mutations. The results reveal that the two mutations enhance the rigidity of P5CR structure, especially that of cofactor binding site, which could result in decreased kinetics of cofactor entrance and egress. Comparison between the structural properties of the WT and mutants during MD simulations shows that the enhanced rigidity of mutants results most likely from the increased number of inter-atomic interactions and the decreased number of dynamic hydrogen bonds. Our study provides novel insight into the deleterious effects of the R119G and G206W mutations on P5CR, and sheds light on the mechanisms by which these mutations mediate Cutis Laxa.

show abstract

Probing the Effect of the Non-Active-Site Mutation Y229W in New Delhi Metallo-β-lactamase-1 by Site-Directed Mutagenesis, Kinetic Studies, and Molecular Dynamics Simulations

Cited by 28 publications

References 36 publications

Modified Deacetylcephalosporin C Synthase for the Biotransformation of Semisynthetic Cephalosporins

Modified Deacetylcephalosporin C Synthase for the Biotransformation of Semisynthetic Cephalosporins

Role of Non-Active-Site Residue Trp-93 in the Function and Stability of New Delhi Metallo-β-Lactamase 1

In silico screening, molecular docking, and molecular dynamics studies of SNP-derived human P5CR mutants

Contact Info

Product

Resources

About