Biology subjects

Barber-Zucker, S.

Publications and source records attributed to Barber-Zucker, S..

5 recordsLinked to original sources

bioRxiv2022

Designed high-redox potential laccases exhibit high functional diversity

White-rot fungi secrete an impressive repertoire of high-redox potential laccases (HRPLs) and peroxidases for efficient oxidation and utilization of lignin. Laccases are attractive enzymes for green-chemistry applications due to their broad substrate range and low environmental impact. Since expression of functional recombinant HRPLs is challenging, however, iterative directed evolution protocols have been applied to improve their expression, activity and stability. We implement a rational, stabilize-and-diversify strategy to two HRPLs that we could not functionally express: first, we use the PROSS stability-design algorithm to allow functional expression in yeast. Second, we use the stabilized enzymes as starting points for FuncLib active-site design to improve their activity and substrate diversity. Four of the FuncLib designed HRPLs and their PROSS progenitor exhibit substantial diversity in reactivity profiles against high-redox potential substrates, including lignin monomers. Combinations of 3-4 subtle mutations that change the polarity, solvation and sterics of the substrate-oxidation site result in orders of magnitude changes in reactivity profiles. These stable and versatile HRPLs are a step towards the generation of an effective lignin-degrading consortium of enzymes that can be secreted from yeast. More broadly, the stabilize-and-diversify strategy can be applied to other challenging enzyme families to study and expand the utility of natural enzymes.

biochemistry↗

bioRxiv2021

Stable and functionally diverse versatile peroxidases by computational design directly from sequence

White-rot fungi secrete a repertoire of high-redox potential oxidoreductases to efficiently decompose lignin. Of these enzymes, versatile peroxidases (VPs) are the most promiscuous biocatalysts. VPs are attractive enzymes for research and industrial use, but their recombinant production is extremely challenging. To date, only a single VP has been structurally characterized and optimized for recombinant functional expression, stability and activity. Computational enzyme optimization methods can be applied to many enzymes in parallel, but they require accurate structures. Here, we demonstrate that model structures computed by deep-learning based ab initio structure prediction methods are reliable starting points for one-shot PROSS stability-design calculations. Four designed VPs encoding as many as 43 mutations relative to the wild type enzymes are functionally expressed in yeast whereas their wild type parents are not. Three of these designs exhibit substantial and useful diversity in reactivity profile and tolerance to environmental conditions. The reliability of the new generation of structure predictors and design methods increases the scale and scope of computational enzyme optimization, enabling efficient discovery and exploitation of the functional diversity in natural enzyme families.

biochemistry↗

bioRxiv2020

The metal binding site composition of the cation diffusion facilitator protein MamM cytoplasmic domain impacts its metal responsivity

The cation diffusion facilitator (CDF) is a conserved family of divalent d-block metal cation transporters that extrude these cations selectively from the cytoplasm. CDF proteins are composed of two domains: the transmembrane domain, through which the cations are transported, and a regulatory cytoplasmic C-terminal domain (CTD). Metal binding to the CTD leads to its tighter conformation, and this sequentially promotes conformational change of the transmembrane domain which allows the actual transport of specific metal cations. It was recently shown that the magnetotactic bacterial CDF protein MamM CTD has a role in metal selectivity, as binding of different metal cations exhibits distinctive affinities and conformations. It is yet unclear whether the composition of the CTD binding sites can impact metal selectivity. Here we performed a mutational study of MamM CTD, where we exchanged the metal binding residues with different metal-binding amino acids. Using X-ray crystallography and Trp-fluorescence spectrometry, we studied the impact of the mutations on the CTD conformation in the presence of different metals. Our results reveal that the incorporation of such mutations alters the domain response to metals in vitro, as mutant forms of the CTD bind metals differently in terms of the composition of the binding sites and the CTD conformation. CoordinatesMamM CTD structures have been deposited in the Protein Data Bank under the following accession codes: 6H5V, 6H5M, 6H5U, 6H8G, 6HAO, 6H88, 6H87, 6H8A, 6H89, 6H8D, 6H5K, 6H9Q, 6H84, 6H83, 6HA2, 6H8I, 6H9T, 6H81, 6HAN, 6H85, 6H9P, 6HHS.

biophysics↗

bioRxiv2020

The cation diffusion facilitator protein MamM's cytoplasmic domain exhibits metal-type dependent binding modes and discriminates against Mn2+

Cation diffusion facilitator (CDF) proteins are a conserved family of divalent transition metal cation transporters. CDF proteins are usually composed of two domains: the transmembrane domain (TMD), in which the metal cations are transported through, and a regulatory cytoplasmic C-terminal domain (CTD). Each CDF protein transports either one specific metal, or multiple metals, from the cytoplasm. Here, the model CDF protein MamM, from magnetotactic bacteria, was used to probe the role of the CTD in metal selectivity. Using a combination of biophysical and structural approaches, the binding of different metals to MamM CTD was characterized. Results reveal that different metals bind distinctively to MamM CTD in terms of; their binding sites, thermodynamics and binding-dependent conformation, both in crystal form and in solution. Furthermore, the results indicate that the CTD discriminates against Mn2+ and provides the first direct evidence that CDF CTDs play a role in metal selectivity.

biophysics↗

bioRxiv2017

Transition metal binding selectivity in proteins and its correlation with the phylogenomic classification of the cation diffusion facilitator protein family

Divalent d-block metal cations (DDMCs), such as Fe, Zn and Mn, participate in many biological processes. Understanding how specific DDMCs are transported to and within the cell and what controls their binding selectivity to different proteins is crucial for defining the mechanisms of metalloproteins. To better understand such processes, we scanned the RCSB Protein Data Bank, performed a de novo structural-based comprehensive analysis of seven DDMCs and found their amino acid binding and coordination geometry propensities. We then utilized these results to characterize the correlation between metal selectivity, specific binding site composition and phylogenetic classification of the cation diffusion facilitator (CDF) protein family, a family of DDMC transporters found throughout evolution and sharing a conserved structure, yet with different members displaying distinct metal selectivity. Our analysis shows that DDMCs differ, at times significantly, in terms of their binding propensities, and that in each CDF clade, the metal selectivity-related binding site has a unique and conserved sequence signature. However, only limited correlation exists between the composition of the DDMC binding site in each clade and the metal selectivity shown by its proteins.

biochemistry↗