Signal Peptides Generated by Attention-Based Neural Networks.

Short (15-30 residue) chains of amino acids at the amino termini of expressed proteins known as signal peptides (SPs) specify secretion in living cells. We trained an attention-based neural network, the Transformer model, on data from all available organisms in Swiss-Prot to generate SP sequences. Experimental testing demonstrates that the model-generated SPs are functional: when appended to enzymes expressed in an industrial Bacillus subtilis strain, the SPs lead to secreted activity that is competitive with industrially used SPs. Additionally, the model-generated SPs are diverse in sequence, sharing as little as 58% sequence identity to the closest known native signal peptide and 73% ± 9% on average.

The use of clade-specific PCR assays to identify novel nitrilase genes from environmental isolates.

Nitrilase enzymes (EC 3.5.5.1) are responsible for the direct hydration of nitriles to their corresponding carboxylic acids and ammonia. The utilization of nitrilase enzymes in biocatalysis toward bio-pharmaceuticals and industrial applications facilitates the move towards green chemistry. The body of research presented describes a novel clade-specific touchdown PCR protocol for the detection of novel nitrilase genes. The presented study identified partial sequences of 15 novel nitrilase genes across 7 genera, with partial DNA sequence homology (%) displayed across an additional 16 genera. This research will prove valuable in the screening of microorganisms for the identification of novel clade-specific nitrilase genes, with predicted enantioselective profiles as determined by their clade characterizations.

Circular permutation provides an evolutionary link between two families of calcium-dependent carbohydrate binding modules.

The microbial deconstruction of the plant cell wall is a critical biological process, which also provides important substrates for environmentally sustainable industries. Enzymes that hydrolyze the plant cell wall generally contain non-catalytic carbohydrate binding modules (CBMs) that contribute to plant cell wall degradation. Here we report the biochemical properties and crystal structure of a family of CBMs (CBM60) that are located in xylanases. Uniquely, the proteins display broad ligand specificity, targeting xylans, galactans, and cellulose. Some of the CBM60s display enhanced affinity for their ligands through avidity effects mediated by protein dimerization. The crystal structure of vCBM60, displays a ß-sandwich with the ligand binding site comprising a broad cleft formed by the loops connecting the two ß-sheets. Ligand recognition at site 1 is, exclusively, through hydrophobic interactions, whereas binding at site 2 is conferred by polar interactions between a protein-bound calcium and the O2 and O3 of the sugar. The observation, that ligand recognition at site 2 requires only a ß-linked sugar that contains equatorial hydroxyls at C2 and C3, explains the broad ligand specificity displayed by vCBM60. The ligand-binding apparatus of vCBM60 displays remarkable structural conservation with a family 36 CBM (CBM36); however, the residues that contribute to carbohydrate recognition are derived from different regions of the two proteins. Three-dimensional structure-based sequence alignments reveal that CBM36 and CBM60 are related by circular permutation. The biological and evolutionary significance of the mechanism of ligand recognition displayed by family 60 CBMs is discussed.

Fully orthogonally protected 2-deoxystreptamine from kanamycin.

A fully orthogonally protected and enantiopure 2-deoxystreptamine derivative is prepared in a few straightforward steps from commercially available kanamycin. Resolution of a sterically hindered diacetate was effected by a Verenium esterase and was followed by a chemoselective Staudinger reduction-acylation protocol.

Engineering hyperthermostability into a GH11 xylanase is mediated by subtle changes to protein structure.

Understanding the structural basis for protein thermostability is of considerable biological and biotechnological importance as exemplified by the industrial use of xylanases at elevated temperatures in the paper pulp and animal feed sectors. Here we have used directed protein evolution to generate hyperthermostable variants of a thermophilic GH11 xylanase, EvXyn11. The Gene Site Saturation Mutagenesis (GSSM) methodology employed assesses the influence on thermostability of all possible amino acid substitutions at each position in the primary structure of the target protein. The 15 most thermostable mutants, which generally clustered in the N-terminal region of the enzyme, had melting temperatures (Tm) 1-8 degrees C higher than the parent protein. Screening of a combinatorial library of the single mutants identified a hyperthermostable variant, EvXyn11TS, containing seven mutations. EvXyn11TS had a Tm approximately 25 degrees C higher than the parent enzyme while displaying catalytic properties that were similar to EvXyn11. The crystal structures of EvXyn11 and EvXyn11TS revealed an absence of substantial changes to identifiable intramolecular interactions. The only explicable mutations are T13F, which increases hydrophobic interactions, and S9P that apparently locks the conformation of a surface loop. This report shows that the molecular basis for the increased thermostability is extraordinarily subtle and points to the requirement for new tools to interrogate protein folding at non-ambient temperatures.

Understanding the structural basis for substrate and inhibitor recognition in eukaryotic GH11 xylanases.

Endo-beta1,4-xylanases (xylanases) hydrolyse the beta1,4 glycosidic bonds in the backbone of xylan. Although xylanases from glycoside hydrolase family 11 (GH11) have been extensively studied, several issues remain unresolved. Thus, the mechanism by which these enzymes hydrolyse decorated xylans is unclear and the structural basis for the variation in catalytic activity within this family is unknown. Furthermore, the mechanism for the differences in the inhibition of fungal GH11 enzymes by the wheat protein XIP-I remains opaque. To address these issues we report the crystal structure and biochemical properties of the Neocallimastix patriciarum xylanase NpXyn11A, which displays unusually high catalytic activity and is one of the few fungal GH11 proteins not inhibited by XIP-I. Although the structure of NpXyn11A could not be determined in complex with substrates, we have been able to investigate how GH11 enzymes hydrolyse decorated substrates by solving the crystal structure of a second GH11 xylanase, EnXyn11A (encoded by an environmental DNA sample), bound to ferulic acid-1,5-arabinofuranose-alpha1,3-xylotriose (FAX(3)). The crystal structure of the EnXyn11A-FAX(3) complex shows that solvent exposure of the backbone xylose O2 and O3 groups at subsites -3 and +2 allow accommodation of alpha1,2-linked 4-methyl-D-glucuronic acid and L-arabinofuranose side chains. Furthermore, the ferulated arabinofuranose side chain makes hydrogen bonds and hydrophobic interactions at the +2 subsite, indicating that the decoration may represent a specificity determinant at this aglycone subsite. The structure of NpXyn11A reveals potential -3 and +3 subsites that are kinetically significant. The extended substrate-binding cleft of NpXyn11A, compared to other GH11 xylanases, may explain why the Neocallimastix enzyme displays unusually high catalytic activity. Finally, the crystal structure of NpXyn11A shows that the resistance of the enzyme to XIP-I is not due solely to insertions in the loop connecting beta strands 11 and 12, as suggested previously, but is highly complex.

Identification of a metagenome-derived esterase with high enantioselectivity in the kinetic resolution of arylaliphatic tertiary alcohols.

35 metagenome-derived esterases bearing a GGG(A)X motif were screened for activity and enantioselectivity in the hydrolysis of a range of tertiary alcohol acetates. Most of the active esterases showed little or no enantioselectivity in the hydrolysis of the terpinyl acetate, linalyl acetate and 3-methylpent-1-yn-3-yl acetate. However, one esterase showed excellent enantioselectivity (E > 100) in the kinetic resolution of 1,1,1-trifluoro-2-phenylbut-3-yn-2-yl acetate as confirmed by a preparative scale reaction.

Epoxide hydrolase-catalyzed enantioselective synthesis of chiral 1,2-diols via desymmetrization of meso-epoxides. lzhao@diversa.com.

The discovery, from nature, of a diverse set of microbial epoxide hydrolases is reported. The utility of a library of epoxide hydrolases in the synthesis of chiral 1,2-diols via desymmetrization of a wide range of meso-epoxides, including cyclic as well as acyclic alkyl- and aryl-substituted substrates, is demonstrated. The chiral (R,R)-diols were furnished with high ee's and yields. The discovery of the first microbial epoxide hydrolases providing access to complementary (S,S)-diols is also described.

Exploring nitrilase sequence space for enantioselective catalysis.

Nitrilases are important in the biosphere as participants in synthesis and degradation pathways for naturally occurring, as well as xenobiotically derived, nitriles. Because of their inherent enantioselectivity, nitrilases are also attractive as mild, selective catalysts for setting chiral centers in fine chemical synthesis. Unfortunately, <20 nitrilases have been reported in the scientific and patent literature, and because of stability or specificity shortcomings, their utility has been largely unrealized. In this study, 137 unique nitrilases, discovered from screening of >600 biotope-specific environmental DNA (eDNA) libraries, were characterized. Using culture-independent means, phylogenetically diverse genomes were captured from entire biotopes, and their genes were expressed heterologously in a common cloning host. Nitrilase genes were targeted in a selection-based expression assay of clonal populations numbering 10(6) to 10(10) members per eDNA library. A phylogenetic analysis of the novel sequences discovered revealed the presence of at least five major sequence clades within the nitrilase subfamily. Using three nitrile substrates targeted for their potential in chiral pharmaceutical synthesis, the enzymes were characterized for substrate specificity and stereospecificity. A number of important correlations were found between sequence clades and the selective properties of these nitrilases. These enzymes, discovered using a high-throughput, culture-independent method, provide a catalytic toolbox for enantiospecific synthesis of a variety of carboxylic acid derivatives, as well as an intriguing library for evolutionary and structural analyses.

An enzyme library approach to biocatalysis: development of nitrilases for enantioselective production of carboxylic acid derivatives.

The discovery, from Nature, of a large and diverse set of nitrilases is reported. The utility of this nitrilase library for identifying enzymes that catalyze efficient production of valuable hydroxy carboxylic acid derivatives is demonstrated. Unprecedented enantioselectivity and substrate scope are highlighted for three newly discovered and distinct nitrilases. For example, a wide array of (R)-mandelic acid derivatives and analogues were produced with high rates, yields, and enantiomeric excesses (95-99% ee). We also have found nitrilases that provide direct access to (S)-phenyllactic acid and other aryllactic acid derivatives, again with high yields and enantioselectivities. Finally, different nitrilases have been discovered that catalyze enantiotopic hydrolysis of 3-hydroxyglutaronitrile to afford either enantiomer of 4-cyano-3-hydroxybutyric acid with high enantiomeric excesses (>95% ee). The first enzymes are reported that effect this transformation to furnish the (R)-4-cyano-3-hydroxybutyric acid which is a precursor to the blockbuster drug Lipitor.