Biology subjects

Hollfelder, F.

Publications and source records attributed to Hollfelder, F..

39 records · Page 3Linked to original sources

bioRxiv2019

Access to unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis

Insertions and deletions (InDels) are frequently observed in natural protein evolution, yet their potential remains untapped in laboratory evolution. Here we introduce a transposon mutagenesis approach (TRIAD) to generate libraries of random variants with short in-frame InDels, and screen TRIAD libraries to evolve a promiscuous arylesterase activity in a phosphotriesterase. The evolution exhibits features that are distinct from previous point mutagenesis campaigns: while the average activity of TRIAD variants is more deleterious, a larger proportion has successfully adapted for the new activity, exhibiting different functional profiles: (i) both strong and weak trade-off in original vs promiscuous activity are observed; (ii) trade-off is more severe (10- to 20-fold increased kcat/KM in arylesterase with [~]100-fold decreases in the original phosphotriesterase activity) and (iii) improvements show up in kcat rather than KM, suggesting novel adaptive solution. These distinct features make TRIAD an alternative to widely used point mutagenesis, providing access to functional innovations and traversing unexplored fitness landscape regions.

biochemistry↗

bioRxiv2018

High-Throughput, Lysis-free Screening for sulfatase Activity Using Escherichia coli Autodisplay in Microdroplets

Directed evolution of enzymes toward improved catalytic performance has become a powerful tool in protein engineering. To be effective, a directed evolution campaign requires the use of high-throughput screening. In this study we describe the development of a high-throughput lysis-free procedure to screen for improved sulfatase activity by combining microdroplet-based single-variant activity sorting with E. coli autodisplay. For the first step in a 4-step screening procedure we quantitatively screened >105 variants of the homodimeric arylsulfatase from Silicibacter pomeroyi (SpAS1), displayed on the E. coli cell surface, for improved sulfatase activity using fluorescence activated droplet sorting. Display of the sulfatase variants on living E. coli cells ensured the continuous linkage of genotype and phenotype during droplet sorting and allowed for direct recovery by simple regrowth of the sorted cells. The use of autodisplay on living cells simplified and reduced the degree of liquid handling during all steps in the screening procedure to the single event of simply mixing substrate and cells. The percentage of apparent improved variants was enriched >10-fold as a result of droplet sorting. We ultimately identified 25 SpAS1-variants with improved performance toward 4-nitrophenyl sulfate (up to 6.2-fold) and/or fluorescein disulfate (up to 30-fold). In SpAS1 variants with improved performance toward the bulky fluorescein disulfate, many of the beneficial mutations occur in residues that form hydrogen bonds between -helices in the C-terminal oligomerization region, suggesting a non-trivial, previously unknown role for the dimer interface in shaping the substrate binding site of SpAS1.\n\nTable of contents graphic\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC=\"FIGDIR/small/479162v2_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (29K):\norg.highwire.dtl.DTLVardef@1f9211aorg.highwire.dtl.DTLVardef@ec3b2dorg.highwire.dtl.DTLVardef@1214667org.highwire.dtl.DTLVardef@1952a50_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2018

Transition State Interactions in a Promiscuous Enzyme: Sulfate and Phosphate Monoester Hydrolysis by Pseudomonas aeruginosa Arylsulfatase

Pseudomonas aeruginosa arylsulfatase (PAS) hydrolyses sulfate and, promiscuously, phosphate monoesters. Enzyme-catalyzed sulfate transfer is crucial to a wide variety of biological processes, but detailed studies of the mechanistic contributions to its catalysis are lacking. We present an investigation based on linear free energy relationships (LFERs) and kinetic isotope effects (KIEs) of PAS and active site mutants that suggest a key role for leaving group (LG) stabilization. In LFERs wild type PAS has a much less negative Br0nsted coefficient ({beta}leaving groupobs-Enz = -0.33) than the uncatalyzed reaction ({beta}leavingroupobs = -1.81). This situation is diminished when cationic active site groups are exchanged for alanine. The considerable degree of bond breaking during the TS is evidenced by an 18Obridge KIE of 1.0088. LFER and KIE data for several active site mutants point to leaving group stabilization by active-site lysine K375, in cooperation with histidine H211. 15N KIEs combined with an increased sensitivity to leaving group ability of the sulfatase activity in neat D2O ({Delta}{beta}leaving groupH-D = +0.06) suggest that the mechanism for S-Obridge bond fission shifts, with decreasing leaving group ability, from charge compensation via Lewis acid interactions towards direct proton donation. 18Ononbridge KIEs indicate that the TS for PAS-catalyzed sulfate monoester hydrolysis has a significantly more associative character compared to the uncatalyzed reaction, while PAS-catalyzed phosphate monoester hydrolysis does not show this shift. This difference in enzyme-catalyzed TSs appears to be the major factor favoring specificity toward sulfate over phosphate in this promiscuous hydrolase, since other features are either too similar (uncatalyzed TS) or inherently favor phosphate (charge).

biochemistry↗