Engineering Biocatalysts for the C-H Activation of Fatty Acids by Ancestral Sequence Reconstruction.

Selective, one-step C-H activation of fatty acids from biomass is an attractive concept in sustainable chemistry. Biocatalysis has shown promise for generating high-value hydroxy acids, but to date enzyme discovery has relied on laborious screening and produced limited hits, which predominantly oxidise the subterminal positions of fatty acids. Herein we show that ancestral sequence reconstruction (ASR) is an effective tool to explore the sequence-activity landscape of a family of multidomain, self-sufficient P450 monooxygenases. We resurrected 11 catalytically active CYP116B ancestors, each with a unique regioselectivity fingerprint that varied from subterminal in the older ancestors to mid-chain in the lineage leading to the extant, P450-TT. In lineages leading to extant enzymes in thermophiles, thermostability increased from ancestral to extant forms, as expected if thermophily had arisen de novo. Our studies show that ASR can be applied to multidomain enzymes to develop active, self-sufficient monooxygenases as regioselective biocatalysts for fatty acid hydroxylation.

Enzymatic Late-Stage Modifications: Better Late Than Never.

Enzyme catalysis is gaining increasing importance in synthetic chemistry. Nowadays, the growing number of biocatalysts accessible by means of bioinformatics and enzyme engineering opens up an immense variety of selective reactions. Biocatalysis especially provides excellent opportunities for late-stage modification often superior to conventional de novo synthesis. Enzymes have proven to be useful for direct introduction of functional groups into complex scaffolds, as well as for rapid diversification of compound libraries. Particularly important and highly topical are enzyme-catalysed oxyfunctionalisations, halogenations, methylations, reductions, and amide bond formations due to the high prevalence of these motifs in pharmaceuticals. This Review gives an overview of the strengths and limitations of enzymatic late-stage modifications using native and engineered enzymes in synthesis while focusing on important examples in drug development.

Corticosteroid monotherapy versus combined cytarabine continuous rate infusion and corticosteroid therapy in dogs with meningoencephalitis of unknown origin: A blinded, randomized, controlled trial.

BACKGROUND: Treatment options available for meningoencephalitis of unknown origin (MUO) in dogs are suboptimal, and currently, no single treatment protocol appears to be superior. OBJECTIVES: Compare neurological deterioration rates at 7 days between dogs with MUO treated with corticosteroids alone or combined with cytosine arabinoside (CA) continuous rate infusion (CRI) and compare clinical deterioration and survival at 30 and 100 days. ANIMALS: Sixty-nine dogs with magnetic resonance imaging (MRI) and cerebrospinal fluid (CSF) features or both compatible with MUO. METHODS: Parallel, blinded, randomized controlled trial. Simple randomization into 2 treatment groups: 4 mg/kg/day prednisolone (or dexamethasone equivalent) for 2 days or 200 mg/m2 CA CRI over 8 hours plus 2 mg/kg/day prednisolone. Blinding of the treatment protocol was carried out using reversible redaction of clinical records, and treatment failure was defined as deterioration of neurological assessment or death. Using intention-to-treat analysis, proportions failing treatment at 7, 30, and 100 days were compared using Fisher's exact test. All-cause mortality at 100 days was compared using Kaplan-Meier survival curves. RESULTS: Thirty-five dogs were allocated to corticosteroid only, and 34 dogs were allocated to combined CA CRI and corticosteroid. Proportions failing treatment at 7, 30, and 100 days were 7/35 (20%), 9/35 (26%), and 15/35 (43%) in the corticosteroid-only group and 8/34 (24%), 11/34 (32%), and 23/34 (68%) in the corticosteroid and CA CRI group. All-cause mortality at 100 days was not significantly different between groups (P = .62). Clinically relevant treatment-related adverse effects were not observed. CONCLUSIONS AND CLINICAL IMPORTANCE: We found no difference in outcome between corticosteroid monotherapy and combined cytarabine CRI and corticosteroid therapy at 7, 30, and 100 days after diagnosis in dogs with MUO.

Biochemical characterization of a glycoside hydrolase family 43 ß-D-galactofuranosidase from the fungus Aspergillus niger.

ß-D-Galactofuranose (Galf) and its polysaccharides are found in bacteria, fungi and protozoa but do not occur in mammalian tissues, and thus represent a specific target for anti-pathogenic drugs. Understanding the enzymatic degradation of these polysaccharides is therefore of great interest, but the identity of fungal enzymes with exclusively galactofuranosidase activity has so far remained elusive. Here we describe the identification and characterization of a galactofuranosidase from the industrially important fungus Aspergillus niger. Analysis of glycoside hydrolase family 43 subfamily 34 (GH43_34) members via conserved unique peptide patterns and phylogeny, revealed the occurrence of distinct clusters and, by comparison with specificities of characterized bacterial members, suggested a basis for prediction of enzyme specificity. Using this rationale, in tandem with molecular docking, we identified a putative ß-D-galactofuranosidase from A. niger which was recombinantly produced in Escherichia coli. The Galf-specific hydrolase, encoded by xynD demonstrates maximum activity at pH 5, 25 °C towards 4-nitrophenyl-ß-galactofuranoside (pNP-ß-Galf), with a Km of 17.9 ± 1.9 mM and Vmax of 70.6 ± 5.3 µM min-1. The characterization of this first fungal GH43 galactofuranosidase offers further molecular insight into the degradation of Galf-containing structures.

Comparison of serum creatine kinase and aspartate aminotransferase activity in dogs with Neospora meningoencephalitis and noninfectious meningoencephalitis.

BACKGROUND: Creatine kinase (CK) and aspartate aminotransferase (AST) activity can be increased with myositis associated with Toxoplasma and Neospora infection in dogs. HYPOTHESIS/OBJECTIVES: Serum activity of CK and AST can be used as a rapid screen for predicting positive serology in meningoencephalitis caused by Toxoplasma gondii or Neospora caninum in dogs compared to dogs with noninfectious meningoencephalitis. ANIMALS: Eighty dogs with meningoencephalitis based on magnetic resonance imaging and cerebrospinal fluid analysis. METHODS: Retrospective case-control study. Serological cutoffs (≥1:800 immunofluorescence for Neospora and ≥1:400 IgG or ≥1:64 IgM or both for Toxoplasma) categorized dogs as infected (n = 21, all neosporosis) or noninfected (n = 59). Activities of CK and AST between infected and noninfected groups were compared using a Mann-Whitney U test and receiver operating characteristic curve analysis. RESULTS: No dogs were diagnosed with toxoplasmosis. Serum CK and AST activities were significantly increased (P < .001) in dogs with positive serology for Neospora (CK: median, 1334 U/L; range, 281-3633 U/L and AST: median, 124 U/L; range, 59-333 U/L) compared to noninfectious cases (CK: median, 215 U/L; range, 69-683 U/L and AST: median, 36 U/L; range, 19-139 U/L). A CK cutoff of 485 U/L had 95.24% sensitivity and 96.61% specificity with a negative predicative value of >99%. An AST cutoff of 57 U/L had 94.44% sensitivity and 85.71% specificity with an estimated negative predicative value of 99%. CONCLUSIONS AND CLINICAL IMPORTANCE: High serum CK and AST activity can increase suspicion for neosporosis while awaiting serological tests for dogs with meningoencephalitis.