Purification, crystallization and preliminary X-ray analysis of the lytic transglycosylase MltA from Escherichia coli

The lytic transglycosylase MltA from Escherichia coli with its membrane anchor and signal sequence deleted has been purified to homogeneity by means of cation-exchange chromatography. The enzyme was crystallized using the hanging-drop vapour-diffusion method. The crystals belong to space group P3(1)21 or P3(2)21, with unit-cell parameters a=b=103.70, c=109.84 Angstrom and one molecule per asymmetric unit. Crystals diffract to 2.2 Angstrom resolution on a synchrotron-radiation source

Using Mutability Landscapes To Guide Enzyme Thermostabilization

Thermostabilizing enzymes while retaining their activity and enantioselectivity for applied biocatalysis is an important topic in protein engineering. Rational and computational design strategies as well as directed evolution have been used successfully to thermostabilize enzymes. Herein, we describe an alternative mutability-landscape approach that identified three single mutations (R11Y, R11I and A33D) within the enzyme 4-oxalocrotonate tautomerase (4-OT), which has potential as a biocatalyst for pharmaceutical synthesis, that gave rise to significant increases in apparent melting temperature Tm (up to 20 °C) and in half-life at 80 °C (up to 111-fold). Introduction of these beneficial mutations in an enantioselective but thermolabile 4-OT variant (M45Y/F50A) afforded improved triple-mutant enzyme variants showing an up to 39 °C increase in Tm value, with no reduction in catalytic activity or enantioselectivity. This study illustrates the power of mutability-landscape-guided protein engineering for thermostabilizing enzymes

Gene Fusion and Directed Evolution to Break Structural Symmetry and Boost Catalysis by an Oligomeric C‐C Bond‐Forming Enzyme

Gene duplication and fusion are among the primary natural processes that generate new proteins from simpler ancestors. Here we adopted this strategy to evolve a promiscuous homohexameric 4-oxalocrotonate tautomerase (4-OT) into an efficient biocatalyst for enantioselective Michael reactions. We first designed a tandem-fused 4-OT to allow independent sequence diversification of adjacent subunits by directed evolution. This fused 4-OT was then subjected to eleven rounds of directed evolution to give variant 4-OT(F11), which showed an up to 320-fold enhanced activity for the Michael addition of nitromethane to cinnamaldehydes. Crystallographic analysis revealed that 4-OT(F11) has an unusual asymmetric trimeric architecture in which one of the monomers is flipped 180° relative to the others. This gene duplication and fusion strategy to break structural symmetry is likely to become an indispensable asset of the enzyme engineering toolbox, finding wide use in engineering oligomeric proteins

Directed Evolution of a Designer Enzyme Featuring an Unnatural Catalytic Amino Acid

The impressive rate accelerations that enzymes display in nature often result from boosting the inherent catalytic activities of side chains by their precise positioning inside a protein binding pocket. Such fine‐tuning is also possible for catalytic unnatural amino acids. Specifically, the directed evolution of a recently described designer enzyme, which utilizes an aniline side chain to promote a model hydrazone formation reaction, is reported. Consecutive rounds of directed evolution identified several mutations in the promiscuous binding pocket, in which the unnatural amino acid is embedded in the starting catalyst. When combined, these mutations boost the turnover frequency (kcat) of the designer enzyme by almost 100‐fold. This results from strengthening the catalytic contribution of the unnatural amino acid, as the engineered designer enzymes outperform variants, in which the aniline side chain is replaced with a catalytically inactive tyrosine residue, by more than 200‐fold

The Role of Tryptophan in π Interactions in Proteins:An Experimental Approach

In proteins, the amino acids Phe, Tyr, and especially Trp are frequently involved in π interactions such as π-π, cation-π, and CH-π bonds. These interactions are often crucial for protein structure and protein-ligand binding. A powerful means to study these interactions is progressive fluorination of these aromatic residues to modulate the electrostatic component of the interaction. However, to date no protein expression platform is available to produce milligram amounts of proteins labeled with such fluorinated amino acids. Here, we present a Lactococcus lactis Trp auxotroph-based expression system for efficient incorporation (≥95%) of mono-, di-, tri-, and tetrafluorinated, as well as a methylated Trp analog. As a model protein we have chosen LmrR, a dimeric multidrug transcriptional repressor protein from L. lactis. LmrR binds aromatic drugs, like daunomycin and riboflavin, between Trp96 and Trp96' in the dimer interface. Progressive fluorination of Trp96 decreased the affinity for the drugs 6- to 70-fold, clearly establishing the importance of electrostatic π-π interactions for drug binding. Presteady state kinetic data of the LmrR-drug interaction support the enthalpic nature of the interaction, while high resolution crystal structures of the labeled protein-drug complexes provide for the first time a structural view of the progressive fluorination approach. The L. lactis expression system was also used to study the role of Trp68 in the binding of riboflavin by the membrane-bound riboflavin transport protein RibU from L. lactis. Progressive fluorination of Trp68 revealed a strong electrostatic component that contributed 15-20% to the total riboflavin-RibU binding energy

Characterization of cytochrome P450 CYP109E1 from Bacillus megaterium as a novel vitamin D3 hydroxylase

In this study the ability of CYP109E1 from Bacillus megaterium to metabolize vitamin D3 (VD3) was investigated. In an in vitro system using bovine adrenodoxin reductase (AdR) and adrenodoxin (Adx4-108), VD3 was converted by CYP109E1 into several products. Furthermore, a whole-cell system in B. megaterium MS941 was established. The new system showed a conversion of 95% after 24h. By NMR analysis it was found that CYP109E1 catalyzes hydroxylation of VD3 at carbons C-24 and C-25, resulting in the formation of 24(S)-hydroxyvitamin D3 (24S(OH)VD3), 25-hydroxyvitamin D3 (25(OH)VD3) and 24S,25-dihydroxyvitamin D3 (24S,25(OH)2VD3). Through time dependent whole-cell conversion of VD3, we identified that the formation of 24S,25(OH)2VD3 by CYP109E1 is derived from VD3 via the intermediate 24S(OH)VD3. Moreover, using docking analysis and site-directed mutagenesis, we identified important active site residues capable of determining substrate specificity and regio-selectivity. HPLC analysis of the whole-cell conversion with the I85A-mutant revealed an increased selectivity towards 25-hydroxylation of VD3 compared with the wild type activity, resulting in an approximately 2-fold increase of 25(OH)VD3 production (45mgl(-1)day(-1)) compared to wild type (24.5mgl(-1)day(-1))

Engineered C–N Lyase:Enantioselective Synthesis of Chiral Synthons for Artificial Dipeptide Sweeteners Catalyzed by an Engineered C-N Lyase

Aspartic acid derivatives with branched N‐alkyl or N‐arylalkyl substituents are valuable precursors to artificial dipeptide sweeteners such as neotame and advantame. The development of a biocatalyst to synthesize these compounds in a single asymmetric step is an as yet unmet challenge. Reported here is an enantioselective biocatalytic synthesis of various difficult N‐substituted aspartic acids, including N‐(3,3‐dimethylbutyl)‐l‐aspartic acid and N‐[3‐(3‐hydroxy‐4‐methoxyphenyl)propyl]‐l‐aspartic acid, precursors to neotame and advantame, respectively, using an engineered variant of ethylenediamine‐N,N′‐disuccinic acid (EDDS) lyase from Chelativorans sp. BNC1. This engineered C–N lyase (mutant D290M/Y320M) displayed a remarkable 1140‐fold increase in activity for the selective hydroamination of fumarate compared to that of the wild‐type enzyme. These results present new opportunities to develop practical multienzymatic processes for the more sustainable and step‐economic synthesis of an important class of food additives