| 1. |
- Corbella Morató, Marina, et al.
(författare)
-
Loop dynamics and the evolution of enzyme activity
- 2023
-
Ingår i: Nature Reviews Chemistry. - : Springer Nature. - 2397-3358. ; 7:8, s. 536-547
-
Forskningsöversikt (refereegranskat)abstract
- In the early 2000s, Tawfik presented his 'New View' on enzyme evolu-tion, highlighting the role of conformational plasticity in expanding the functional diversity of limited repertoires of sequences. This view is gaining increasing traction with increasing evidence of the importance of conformational dynamics in both natural and laboratory evolution of enzymes. The past years have seen several elegant examples of harnessing conformational (particularly loop) dynamics to successfully manipulate protein function. This Review revisits flexible loops as critical participants in regulating enzyme activity. We showcase several systems of particular interest: triosephosphate isomerase barrel proteins, protein tyrosine phosphatases and beta-lactamases, while briefly discussing other systems in which loop dynamics are important for selectivity and turnover. We then discuss the implications for engineering, presenting examples of successful loop manipulation in either improving catalytic efficiency, or changing selectivity completely. Overall, it is becoming clearer that mimicking nature by manipulating the conformational dynamics of key protein loops is a powerful method of tailoring enzyme activity, without needing to target active-site residues. [GRAPHICS] .
|
|
| 2. |
- Corbella Morató, Marina, et al.
(författare)
-
The N-terminal Helix-Turn-Helix Motif of Transcription Factors MarA and Rob Drives DNA Recognition
- 2021
-
Ingår i: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 125:25, s. 6791-6806
-
Tidskriftsartikel (refereegranskat)abstract
- DNA-binding proteins play an important role in gene regulation and cellular function. The transcription factors MarA and Rob are two homologous members of the AraC/XylS family that regulate multidrug resistance. They share a common DNA-binding domain, and Rob possesses an additional C-terminal domain that permits binding of low-molecular weight effectors. Both proteins possess two helix-turn-helix (HTH) motifs capable of binding DNA; however, while MarA interacts with its promoter through both HTH-motifs, prior studies indicate that Rob binding to DNA via a single HTH-motif is sufficient for tight binding. In the present work, we perform microsecond time scale all-atom simulations of the binding of both transcription factors to different DNA sequences to understand the determinants of DNA recognition and binding. Our simulations characterize sequence-dependent changes in dynamical behavior upon DNA binding, showcasing the role of Arg40 of the N-terminal HTH-motif in allowing for specific tight binding. Finally, our simulations demonstrate that an acidic C-terminal loop of Rob can control the DNA binding mode, facilitating interconversion between the distinct DNA binding modes observed in MarA and Rob. In doing so, we provide detailed molecular insight into DNA binding and recognition by these proteins, which in turn is an important step toward the efficient design of antivirulence agents that target these proteins.
|
|
| 3. |
- Enugala, Thilak Reddy, et al.
(författare)
-
The Role of Substrate-Coenzyme Crosstalk in Determining Turnover Rates in Rhodococcus ruber Alcohol Dehydrogenase
- 2020
-
Ingår i: ACS Catalysis. - : American Chemical Society (ACS). - 2155-5435 .- 2155-5435. ; 10:16, s. 9115-9128
-
Tidskriftsartikel (refereegranskat)abstract
- Eight related alcohol dehydrogenases that had been originally isolated by laboratory evolution of ADH-A from Rhodococcus ruber DSM44541 for modified substrate scopes, were together with their parent wild-type, subjected to biochemical characterization of possible activities with a panel of chiral alcohols and pro-chiral ketones. Determinations of rates of catalyzed alcohol oxidations and ketone reductions, and of cofactor release, pointed out to the role of a W295A substitution as being decisive in steering enantioselectivity in the oxidation of arylated 1-methyl substituted alcohols. Molecular dynamics simulations of enzyme-substrate interactions in the Michaelis complexes of wild-type and a Y294F/W295A double mutant could rationalize the experimentally observed shift in enantioselectivity and differences in catalytic activity with 4-phenyl-2-butanol. Finally, we present herein evidence for apparent inter-dependency between substrate/product and the cofactor in the ternary complex, that directly affects the NADH dissociation rates, and thus that this substrate-coenzyme crosstalk plays a direct role in determining the turnover rates.
|
|
| 4. |
- Pinto, Gaspar P., et al.
(författare)
-
Exploiting enzyme evolution for computational protein design
- 2022
-
Ingår i: TIBS -Trends in Biochemical Sciences. Regular ed.. - : Elsevier. - 0968-0004 .- 1362-4326. ; 47:5, s. 375-389
-
Forskningsöversikt (refereegranskat)abstract
- Recent years have seen an explosion of interest in understanding the physicochemical parameters that shape enzyme evolution, as well as substantial advances in computational enzyme design. This review discusses three areas where evolutionary information can be used as part of the design process: (i) using ancestral sequence reconstruction (ASR) to generate new starting points for enzyme design efforts; (ii) learning from how nature uses conformational dynamics in enzyme evolution to mimic this process in silico; and (iii) modular design of enzymes from smaller fragments, again mimicking the process by which nature appears to create new protein folds. Using showcase examples, we highlight the importance of incorporating evolutionary information to continue to push forward the boundaries of enzyme design studies.
|
|
| 5. |
- Romero-Rivera, Adrian, et al.
(författare)
-
Complex Loop Dynamics Underpin Activity, Specificity, and Evolvability in the (beta alpha)(8) Barrel Enzymes of Histidine and Tryptophan Biosynthesi
- 2022
-
Ingår i: JACS Au. - : American Chemical Society (ACS). - 2691-3704. ; 2:4, s. 943-960
-
Tidskriftsartikel (refereegranskat)abstract
- Enzymes are conformationally dynamic, and their dynamical properties play an important role in regulating their specificity and evolvability. In this context, substantial attention has been paid to the role of ligand-gated conformational changes in enzyme catalysis; however, such studies have focused on tremendously proficient enzymes such as triosephosphate isomerase and orotidine 5'-monophosphate decarboxylase, where the rapid (mu s timescale) motion of a single loop dominates the transition between catalytically inactive and active conformations. In contrast, the (beta alpha)(8)-barrels of tryptophan and histidine biosynthesis, such as the specialist isomerase enzymes HisA and TrpF, and the bifunctional isomerase PriA, are decorated by multiple long loops that undergo conformational transitions on the ms (or slower) timescale. Studying the interdependent motions of multiple slow loops, and their role in catalysis, poses a significant computational challenge. This work combines conventional and enhanced molecular dynamics simulations with empirical valence bond simulations to provide rich details of the conformational behavior of the catalytic loops in HisA, PriA, and TrpF, and the role of their plasticity in facilitating bifunctionality in PriA and evolved HisA variants. In addition, we demonstrate that, similar to other enzymes activated by ligand-gated conformational changes, loops 3 and 4 of HisA and PriA act as gripper loops, facilitating the isomerization of the large bulky substrate ProFAR, albeit now on much slower timescales. This hints at convergent evolution on these different (beta alpha)(8)-barrel scaffolds. Finally, our work reemphasizes the potential of engineering loop dynamics as a tool to artificially manipulate the catalytic repertoire of TIM-barrel proteins.
|
|
| 6. |
- Shen, Ruidan, et al.
(författare)
-
Insights into the importance of WPD-loop sequence for activity and structure in protein tyrosine phosphatases
- 2022
-
Ingår i: Chemical Science. - : Royal Society of Chemistry. - 2041-6520 .- 2041-6539. ; 13:45, s. 13524-13540
-
Tidskriftsartikel (refereegranskat)abstract
- Protein tyrosine phosphatases (PTPs) possess a conserved mobile catalytic loop, the WPD-loop, which brings an aspartic acid into the active site where it acts as an acid/base catalyst. Prior experimental and computational studies, focused on the human enzyme PTP1B and the PTP from Yersinia pestis, YopH, suggested that loop conformational dynamics are important in regulating both catalysis and evolvability. We have generated a chimeric protein in which the WPD-loop of YopH is transposed into PTP1B, and eight chimeras that systematically restored the loop sequence back to native PTP1B. Of these, four chimeras were soluble and were subjected to detailed biochemical and structural characterization, and a computational analysis of their WPD-loop dynamics. The chimeras maintain backbone structural integrity, with somewhat slower rates than either wild-type parent, and show differences in the pH dependency of catalysis, and changes in the effect of Mg2+. The chimeric proteins' WPD-loops differ significantly in their relative stability and rigidity. The time required for interconversion, coupled with electrostatic effects revealed by simulations, likely accounts for the activity differences between chimeras, and relative to the native enzymes. Our results further the understanding of connections between enzyme activity and the dynamics of catalytically important groups, particularly the effects of non-catalytic residues on key conformational equilibria.
|
|
