Biochemical properties of a Pseudomonas aminotransferase involved in caprolactam metabolism

The biodegradation of the nylon-6 precursor caprolactam by a strain of Pseudomonas jessenii proceeds via ATP-dependent hydrolytic ring-opening to 6-aminohexanoate. This non-natural ω-amino acid is converted to 6-oxohexanoic acid by an aminotransferase (PjAT) belonging to the fold type I PLP enzymes. To understand the structural basis of 6-aminohexanoatate conversion, we solved different crystal structures and determined the substrate scope with a range of aliphatic and aromatic amines. Comparison with the homologous aminotransferases from Chromobacterium violaceum (CvAT) and Vibrio fluvialis (VfAT) showed that the PjAT enzyme has the lowest KM values (highest affinity) and highest specificity constant (kcat /KM ) with the caprolactam degradation intermediates 6-aminohexanoate and 6-oxohexanoic acid, in accordance with its proposed in vivo function. Five distinct three-dimensional structures of PjAT were solved by protein crystallography. The structure of the aldimine intermediate formed from 6-aminohexanoate and the PLP cofactor revealed the presence of a narrow hydrophobic substrate-binding tunnel leading to the cofactor and covered by a flexible arginine, which explains the high activity and selectivity of the PjAT with 6-aminohexanoate. The results suggest that the degradation pathway for caprolactam has recruited an aminotransferase that is well adapted to 6-aminohexanoate degradation. This article is protected by copyright. All rights reserved

Catalytic and structural properties of ATP-dependent caprolactamase from Pseudomonas jessenii

Caprolactamase is the first enzyme in the caprolactam degradation pathway of Pseudomonas jessenii. It is composed of two subunits (CapA and CapB) and sequence‐related to other ATP‐dependent enzymes involved in lactam hydrolysis, like 5‐oxoprolinases and hydantoinases. Low sequence similarity also exists with ATP‐dependent acetone‐ and acetophenone carboxylases. The caprolactamase was produced in Escherichia coli, isolated by His‐tag affinity chromatography, and subjected to functional and structural studies. Activity toward caprolactam required ATP and was dependent on the presence of bicarbonate in the assay buffer. The hydrolysis product was identified as 6‐aminocaproic acid. Quantum mechanical modeling indicated that the hydrolysis of caprolactam was highly disfavored (ΔG(0)'= 23 kJ/mol), which explained the ATP dependence. A crystal structure showed that the enzyme exists as an (αβ)(2) tetramer and revealed an ATP‐binding site in CapA and a Zn‐coordinating site in CapB. Mutations in the ATP‐binding site of CapA (D11A and D295A) significantly reduced product formation. Mutants with substitutions in the metal binding site of CapB (D41A, H99A, D101A, and H124A) were inactive and less thermostable than the wild‐type enzyme. These residues proved to be essential for activity and on basis of the experimental findings we propose possible mechanisms for ATP‐dependent lactam hydrolysis

Thermostable D-amino acid decarboxylases derived from Thermotoga maritima diaminopimelate decarboxylase

Diaminopimelate decarboxylases (DAPDCs) are highly selective enzymes that catalyze the common final step in different lysine biosynthetic pathways, i.e. the conversion of meso-diaminopimelate (DAP) to L-lysine. We examined the modification of the substrate specificity of the thermostable decarboxylase from Thermotoga maritima with the aim to introduce activity with 2-aminopimelic acid (2-APA) since its decarboxylation leads to 6-aminocaproic acid (6-ACA), a building block for the synthesis of nylon-6. Structure-based mutagenesis of the distal carboxylate binding site resulted in a set of enzyme variants with new activities toward different D-amino acids. One of the mutants (E315T) had lost most of its activity toward DAP and primarily acted as a 2-APA decarboxylase. We next used computational modeling to explain the observed shift in catalytic activities of the mutants. The results suggest that predictive computational protocols can support the redesign of the catalytic properties of this class of decarboxylating PLP-dependent enzymes

Robust ω-Transaminases by Computational Stabilization of the Subunit Interface

Transaminases are attractive catalysts for the production of enantiopure amines. However, the poor stability of these enzymes often limits their application in biocatalysis. Here, we used a framework for enzyme stability engineering by computational library design (FRESCO) to stabilize the homodimeric PLP fold type I ω-transaminase from Pseudomonas jessenii. A large number of surface-located point mutations and mutations predicted to stabilize the subunit interface were examined. Experimental screening revealed that 10 surface mutations out of 172 tested were indeed stabilizing (6% success), whereas testing 34 interface mutations gave 19 hits (56% success). Both the extent of stabilization and the spatial distribution of stabilizing mutations showed that the subunit interface was critical for stability. After mutations were combined, 2 very stable variants with 4 and 6 mutations were obtained, which in comparison to wild type (Tm app = 62 °C) displayed Tm app values of 80 and 85 °C, respectively. These two variants were also 5-fold more active at their optimum temperatures and tolerated high concentrations of isopropylamine and cosolvents. This allowed conversion of 100 mM acetophenone to (S)-1-phenylethylamine (>99% enantiomeric excess) with high yield (92%, in comparison to 24% with the wild-type transaminase). Crystal structures mostly confirmed the expected structural changes and revealed that the most stabilizing mutation, I154V, featured a rarely described stabilization mechanism: namely, removal of steric strain. The results show that computational interface redesign can be a rapid and powerful strategy for transaminase stabilization

A monomer-trimer model supports intermittent glucagon fibril growth

We investigate in vitro fibrillation kinetics of the hormone peptide glucagon at various concentrations using confocal microscopy and determine the glucagon fibril persistence length 60μm. At all concentrations we observe that periods of individual fibril growth are interrupted by periods of stasis. The growth probability is large at high and low concentrations and is reduced for intermediate glucagon concentrations. To explain this behavior we propose a simple model, where fibrils come in two forms, one built entirely from glucagon monomers and one entirely from glucagon trimers. The opposite building blocks act as fibril growth blockers, and this generic model reproduces experimental behavior well

Metabolism of β-valine via a CoA-dependent ammonia lyase pathway

Pseudomonas species strain SBV1 can rapidly grow on medium containing β-valine as a sole nitrogen source. The tertiary amine feature of β-valine prevents direct deamination reactions catalyzed by aminotransferases, amino acid dehydrogenases, and amino acid oxidases. However, lyase- or aminomutase-mediated conversions would be possible. To identify enzymes involved in the degradation of β-valine, a PsSBV1 gene library was prepared and used to complement the β-valine growth deficiency of a closely related Pseudomonas strain. This resulted in the identification of a gene encoding β-valinyl-coenzyme A ligase (BvaA) and two genes encoding β-valinyl-CoA ammonia lyases (BvaB1 and BvaB2). The BvaA protein demonstrated high sequence identity to several known phenylacetate CoA ligases. Purified BvaA enzyme did not convert phenyl acetic acid but was able to activate β-valine in an adenosine triphosphate (ATP)- and CoA-dependent manner. The substrate range of the enzyme appears to be narrow, converting only β-valine and to a lesser extent, 3-aminobutyrate and β-alanine. Characterization of BvaB1 and BvaB2 revealed that both enzymes were able to deaminate β-valinyl-CoA to produce 3-methylcrotonyl-CoA, a common intermediate in the leucine degradation pathway. Interestingly, BvaB1 and BvaB2 demonstrated no significant sequence identity to known CoA-dependent ammonia lyases, suggesting they belong to a new family of enzymes. BLAST searches revealed that BvaB1 and BvaB2 show high sequence identity to each other and to several enoyl-CoA hydratases, a class of enzymes that catalyze a similar reaction with water instead of amine as the leaving group

GFP fusions of Sec-routed extracellular proteins in Staphylococcus aureus reveal surface-associated coagulase in biofilms

Staphylococcus aureus is a major human pathogen that utilises many surface-associated and secreted proteins to form biofilms and cause disease. However, our understanding of these processes is limited by challenges of using fluores-cent protein reporters in their native environment, because they must be ex-ported and fold correctly to become fluorescent. Here, we demonstrate the feasibility of using the monomeric superfolder GFP (msfGFP) exported from S. aureus. By fusing msfGFP to signal peptides for the Secretory (Sec) and Twin Arginine Translocation (Tat) pathways, the two major secretion pathways in S. aureus, we quantified msfGFP fluorescence in bacterial cultures and cell-free supernatant from the cultures. When fused to a Tat signal peptide, we detect-ed msfGFP fluorescence inside but not outside bacterial cells, indicating a fail-ure to export msfGFP. However, when fused to a Sec signal peptide, msfGFP fluorescence was present outside cells, indicating successful export of the msfGFP in the unfolded state, followed by extracellular folding and maturation to the photoactive state. We applied this strategy to study coagulase (Coa), a secreted protein and a major contributor to the formation of a fibrin network in S. aureus biofilms that protects bacteria from the host immune system and increases attachment to host surfaces. We confirmed that a genomically inte-grated C-terminal fusion of Coa to msfGFP does not impair the activity of Coa or its localisation within the biofilm matrix. Our findings demonstrate that msfGFP is a good candidate fluorescent reporter to consider when studying proteins secreted by the Sec pathway in S. aureus