Biology subjects

Tawfik, D. S.

Publications and source records attributed to Tawfik, D. S..

4 recordsLinked to original sources

bioRxiv2018

Chance and pleiotropy dominate genetic diversity in complex bacterial environments

How does environmental complexity affect the evolution of single genes? Here, we measured the effects of a set of mutants of Bacillus subtilis glutamate dehydrogenase across 19 different environments - from homogenous single cell populations in liquid media to heterogeneous biofilms, plant roots and soil communities. The effects of individual gene mutations on organismal fitness were highly reproducible in liquid cultures. Strikingly, however, 84% of the tested alleles showed opposing fitness effects under different growth conditions (environmental pleiotropy). In biofilms and soil samples, different alleles dominated in parallel replica experiments. Accordingly, we found that in these heterogeneous cell communities the fate of mutations was dictated by a combination of selection and drift. The latter relates to programmed prophage excisions that occurred along biofilm development. Overall, per individual condition, by the combined action of selection, pleiotropy and chance, a wide range of glutamate dehydrogenase mutations persisted and sometimes fixated. However, across longer periods and multiple environments, nearly all this diversity would be lost - indeed, considering all environments and conditions we have tested, wild-type is the fittest allele.

microbiology↗

bioRxiv2017

Rescue of Conformational Dynamics in Enzyme Catalysis by Directed Evolution

Rational design and directed evolution have proved to be successful approaches to increase catalytic efficiencies of both natural and artificial enzymes1-3. However, a comprehensive understanding of how evolution shapes the energy landscape of catalysis remains a fundamental challenge. Protein dynamics is widely recognized as important, but due to the inherent flexibility of biological macromolecules it is often difficult to distinguish which conformational changes are directly related to function. Here, we used directed evolution on an impaired mutant of the human proline isomerase cyclophilin A (CypA) and identify two second-shell mutations that partially restore its catalytic activity. We show both kinetically, using NMR spectroscopy, and structurally, by room-temperature X-ray crystallography, how local perturbations propagate through a large allosteric network to facilitate conformational dynamics. The increased catalysis selected for in the evolutionary screen could be rationalized entirely by accelerated interconversion between the two catalytically essential conformational sub-states, which are both captured in the high-resolution X-ray ensembles at room temperature. Our data provide a glimpse of the evolutionary trajectory of an enzymes energy landscape and shows how subtle changes can fine-tune its function.

biophysics↗

bioRxiv2017

Evolution of chalcone isomerase from a non-catalytic ancestor

The emergence of catalysis in a non-catalytic protein scaffold is a rare, unexplored event. Chalcone isomerase (CHI), a key enzyme in plant flavonoid biosynthesis, is presumed to have evolved from a non-enzymatic ancestor related to the widely-distributed fatty-acid binding proteins (FAPs) and a plant protein family with no isomerase activity (CHILs for \"CHI-like\"). Ancestral inference confirmed that CHI evolved from a protein lacking isomerase activity. We also identified four alternative founder mutations, i.e. mutations that individually instated activity, including a mutation that is not phylogenetically traceable. Despite strong epistasis in other cases of protein evolution, CHIs laboratory reconstructed mutational trajectory shows weak epistasis. Thus, enantioselective CHI activity can readily emerge despite a catalytically inactive starting point. X-ray crystallography, NMR, and MD simulations reveal reshaping of the active site toward a productive substrate-binding mode and repositioning of the catalytic arginine that was inherited from the ancestral fatty-acid binding proteins.

biochemistry↗

bioRxiv2017

DddY is a bacterial dimethylsulfoniopropionate lyase representing a new cupin enzyme superfamily with unknown primary function

Dimethylsulfide (DMS) is released at rates of >107 tons annually and plays a key role in the oceanic sulfur cycle and ecology. Marine bacteria, algae, and possibly other organisms, release DMS via cleavage of dimethylsulfoniopropionate (DMSP). Different genes encoding proteins with DMSP lyase activity are known belonging to different superfamilies and exhibiting highly variable levels of DMSP lyase activity. DddY shows the highest activity among all reported bacterial lyases yet is poorly characterized. Here, we describe the characterization of recombinant DddY is from different marine bacteria. We found that DddY activity demands a transition metal ion cofactor. DddY also shares two sequence motifs with other bacterial lyases assigned as cupin-like enzymes, DddQ, DddL, DddK, and DddW. These cupin motif residues are essential for DddY activity, as for the other cupin DMSP lyases, and all these enzymes are characterized by a common metal-chelator inhibitor (TPEN). Analysis of all sequences carrying these cupin motifs defined a superfamily: Cupin-DLL (DMSP lyases and lyase-like). The DMSP lyase families are sporadically distributed suggesting that DMSP lyases evolved within this superfamily independently along multiple lineages. However, the specific activity levels, genomic context analysis, and systematic profiling of substrate selectivity as described in the accompanying paper, indicate that for only some of these families, most distinctly DddY and DddL, DMSP lyase is the primary, native activity. In other families, foremost DddQ, DMSP lyase seems to be merely a promiscuous activity. The native function of DddQ, and of nearly all members of this newly identified Cupin-DLL superfamily, remains unknown.\n\nAbbreviations\n\nFundingFinancial support by the Estate of Mark Scher, and the Sasson & Marjorie Peress Philanthropic Fund, are gratefully acknowledged. D.S.T. is the Nella and Leon Benoziyo Professor of Biochemistry.

biochemistry↗