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 (ΔG0 '= 23 kJ/mol), which explained the ATP dependence. A crystal structure showed that the enzyme exists as an (αß)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.

Mutations in PMR1 stimulate xylose isomerase activity and anaerobic growth on xylose of engineered Saccharomyces cerevisiae by influencing manganese homeostasis.

Combined overexpression of xylulokinase, pentose-phosphate-pathway enzymes and a heterologous xylose isomerase (XI) is required but insufficient for anaerobic growth of Saccharomyces cerevisiae on d-xylose. Single-step Cas9-assisted implementation of these modifications yielded a yeast strain expressing Piromyces XI that showed fast aerobic growth on d-xylose. However, anaerobic growth required a 12-day adaptation period. Xylose-adapted cultures carried mutations in PMR1, encoding a Golgi Ca2+/Mn2+ ATPase. Deleting PMR1 in the parental XI-expressing strain enabled instantaneous anaerobic growth on d-xylose. In pmr1 strains, intracellular Mn2+ concentrations were much higher than in the parental strain. XI activity assays in cell extracts and reconstitution experiments with purified XI apoenzyme showed superior enzyme kinetics with Mn2+ relative to other divalent metal ions. This study indicates engineering of metal homeostasis as a relevant approach for optimization of metabolic pathways involving metal-dependent enzymes. Specifically, it identifies metal interactions of heterologous XIs as an underexplored aspect of engineering xylose metabolism in yeast.

Enzymatic network for production of ether amines from alcohols.

We constructed an enzymatic network composed of three different enzymes for the synthesis of valuable ether amines. The enzymatic reactions are interconnected to catalyze the oxidation and subsequent transamination of the substrate and to provide cofactor recycling. This allows production of the desired ether amines from the corresponding ether alcohols with inorganic ammonium as the only additional substrate. To examine conversion, individual and overall reaction equilibria were established. Using these data, it was found that the experimentally observed conversions of up to 60% observed for reactions containing 10 mM alcohol and up to 280 mM ammonia corresponded well to predicted conversions. The results indicate that efficient amination can be driven by high concentrations of ammonia and may require improving enzyme robustness for scale-up. Biotechnol. Bioeng. 2016;113: 1853-1861. © 2016 Wiley Periodicals, Inc.

Metabolism of ß-valine via a CoA-dependent ammonia lyase pathway.

Pseudomonas species strain SBV1 can rapidly grow on medium containing ß-valine as a sole nitrogen source. The tertiary amine feature of ß-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 ß-valine, a PsSBV1 gene library was prepared and used to complement the ß-valine growth deficiency of a closely related Pseudomonas strain. This resulted in the identification of a gene encoding ß-valinyl-coenzyme A ligase (BvaA) and two genes encoding ß-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 ß-valine in an adenosine triphosphate (ATP)- and CoA-dependent manner. The substrate range of the enzyme appears to be narrow, converting only ß-valine and to a lesser extent, 3-aminobutyrate and ß-alanine. Characterization of BvaB1 and BvaB2 revealed that both enzymes were able to deaminate ß-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.

Peptide synthesis in neat organic solvents with novel thermostable proteases.

Biocatalytic peptide synthesis will benefit from enzymes that are active at low water levels in organic solvent compositions that allow good substrate and product solubility. To explore the use of proteases from thermophiles for peptide synthesis under such conditions, putative protease genes of the subtilase class were cloned from Thermus aquaticus and Deinococcus geothermalis and expressed in Escherichia coli. The purified enzymes were highly thermostable and catalyzed efficient peptide bond synthesis at 80°C and 60°C in neat acetonitrile with excellent conversion (>90%). The enzymes tolerated high levels of N,N-dimethylformamide (DMF) as a cosolvent (40-50% v/v), which improved substrate solubility and gave good conversion in 5+3 peptide condensation reactions. The results suggest that proteases from thermophiles can be used for peptide synthesis under harsh reaction conditions.

X-ray crystallographic validation of structure predictions used in computational design for protein stabilization.

Protein engineering aimed at enhancing enzyme stability is increasingly supported by computational methods for calculation of mutant folding energies and for the design of disulfide bonds. To examine the accuracy of mutant structure predictions underlying these computational methods, crystal structures of thermostable limonene epoxide hydrolase variants obtained by computational library design were determined. Four different predicted effects indeed contributed to the obtained stabilization: (i) enhanced interactions between a flexible loop close to the N-terminus and the rest of the protein; (ii) improved interactions at the dimer interface; (iii) removal of unsatisfied hydrogen bonding groups; and (iv) introduction of additional positively charged groups at the surface. The structures of an eightfold and an elevenfold mutant showed that most mutations introduced the intended stabilizing interactions, and side-chain conformations were correctly predicted for 72-88% of the point mutations. However, mutations that introduced a disulfide bond in a flexible region had a larger influence on the backbone conformation than predicted. The enzyme active sites were unaltered, in agreement with the observed preservation of catalytic activities. The structures also revealed how a c-Myc tag, which was introduced for facile detection and purification, can reduce access to the active site and thereby lower the catalytic activity. Finally, sequence analysis showed that comprehensive mutant energy calculations discovered stabilizing mutations that are not proposed by the consensus or B-FIT methods.

Stabilization of cyclohexanone monooxygenase by a computationally designed disulfide bond spanning only one residue.

Enzyme stability is an important parameter in biocatalytic applications, and there is a strong need for efficient methods to generate robust enzymes. We investigated whether stabilizing disulfide bonds can be computationally designed based on a model structure. In our approach, unlike in previous disulfide engineering studies, short bonds spanning only a few residues were included. We used cyclohexanone monooxygenase (CHMO), a Baeyer-Villiger monooxygenase (BVMO) from Acinetobacter sp. NCIMB9871 as the target enzyme. This enzyme has been the prototype BVMO for many biocatalytic studies even though it is notoriously labile. After creating a small library of mutant enzymes with introduced cysteine pairs and subsequent screening for improved thermostability, three stabilizing disulfide bonds were identified. The introduced disulfide bonds are all within 12 Å of each other, suggesting this particular region is critical for unfolding. This study shows that stabilizing disulfide bonds do not have to span many residues, as the most stabilizing disulfide bond, L323C-A325C, spans only one residue while it stabilizes the enzyme, as shown by a 6 °C increase in its apparent melting temperature.

Priming ammonia lyases and aminomutases for industrial and therapeutic applications.

Ammonia lyases (AL) and aminomutases (AM) are emerging in green synthetic routes to chiral amines and an AL is being explored as an enzyme therapeutic for treating phenylketonuria and cancer. Although the restricted substrate range of the wild-type enzymes limits their widespread application, the non-reliance on external cofactors and direct functionalization of an olefinic bond make ammonia lyases attractive biocatalysts for use in the synthesis of natural and non-natural amino acids, including ß-amino acids. The approach of combining structure-guided enzyme engineering with efficient mutant library screening has extended the synthetic scope of these enzymes in recent years and has resolved important mechanistic issues for AMs and ALs, including those containing the MIO (4-methylideneimidazole-5-one) internal cofactor.

Purification and use of E. coli peptide deformylase for peptide deprotection in chemoenzymatic peptide synthesis.

Peptide deformylases (PDFs) catalyze the removal of the formyl group from the N-terminal methionine residue in nascent polypeptide chains in prokaryotes. Its deformylation activity makes PDF an attractive candidate for the biocatalytic deprotection of formylated peptides that are used in chemoenzymatic peptide synthesis. For this application it is essential to use PDF preparations that are free of contamination by peptidases that can cleave internal peptide bonds. Therefore, different purification methods were attempted and an industrially applicable purification procedure was developed based on a single anion-exchange chromatography step of an engineered PDF variant that was equipped with an anionic octaglutamate tag. The deformylation activity and stability of the engineered enzyme were similar to those of the wild-type PDF. This purification method furnished a PDF preparation with a 1500-fold decreased level of contamination by amidases and peptidases as compared to cell-free extract. It was shown that the enzyme could be used for deprotection of a formylated dipeptide that was prepared by thermolysin-mediated coupling.

Transformation and biodegradation of 1,2,3-trichloropropane (TCP).

PURPOSE: 1,2,3-Trichloropropane (TCP) is a persistent groundwater pollutant and a suspected human carcinogen. It is also is an industrial chemical waste that has been formed in large amounts during epichlorohydrin manufacture. In view of the spread of TCP via groundwater and its toxicity, there is a need for cheap and efficient technologies for the cleanup of TCP-contaminated sites. In situ or on-site bioremediation of TCP is an option if biodegradation can be achieved and stimulated. This paper presents an overview of methods for the remediation of TCP-contaminated water with an emphasis on the possibilities of biodegradation. CONCLUSIONS: Although TCP is a xenobiotic chlorinated compound of high chemical stability, a number of abiotic and biotic conversions have been demonstrated, including abiotic oxidative conversion in the presence of a strong oxidant and reductive conversion by zero-valent zinc. Biotransformations that have been observed include reductive dechlorination, monooxygenase-mediated cometabolism, and enzymatic hydrolysis. No natural organisms are known that can use TCP as a carbon source for growth under aerobic conditions, but anaerobically TCP may serve as electron acceptor. The application of biodegradation is hindered by low degradation rates and incomplete mineralization. Protein engineering and genetic modification can be used to obtain microorganisms with enhanced TCP degradation potential.

Novel dehalogenase mechanism for 2,3-dichloro-1-propanol utilization in Pseudomonas putida strain MC4.

A Pseudomonas putida strain (MC4) that can utilize 2,3-dichloro-1-propanol (DCP) and several aliphatic haloacids and haloalcohols as sole carbon and energy source for growth was isolated from contaminated soil. Degradation of DCP was found to start with oxidation and concomitant dehalogenation catalyzed by a 72-kDa monomeric protein (DppA) that was isolated from cell lysate. The dppA gene was cloned from a cosmid library and appeared to encode a protein equipped with a signal peptide and that possessed high similarity to quinohemoprotein alcohol dehydrogenases (ADHs), particularly ADH IIB and ADH IIG from Pseudomonas putida HK. This novel dehalogenating dehydrogenase has a broad substrate range, encompassing a number of nonhalogenated alcohols and haloalcohols. With DCP, DppA exhibited a k(cat) of 17 s(-1). (1)H nuclear magnetic resonance experiments indicated that DCP oxidation by DppA in the presence of 2,6-dichlorophenolindophenol (DCPIP) and potassium ferricyanide [K(3)Fe(CN)(6)] yielded 2-chloroacrolein, which was oxidized to 2-chloroacrylic acid.

Export of functional Streptomyces coelicolor alditol oxidase to the periplasm or cell surface of Escherichia coli and its application in whole-cell biocatalysis.

Streptomyces coelicolor A3(2) alditol oxidase (AldO) is a soluble monomeric flavoprotein in which the flavin cofactor is covalently linked to the polypeptide chain. AldO displays high reactivity towards different polyols such as xylitol and sorbitol. These characteristics make AldO industrially relevant, but full biotechnological exploitation of this enzyme is at present restricted by laborious and costly purification steps. To eliminate the need for enzyme purification, this study describes a whole-cell AldO biocatalyst system. To this end, we have directed AldO to the periplasm or cell surface of Escherichia coli. For periplasmic export, AldO was fused to endogenous E. coli signal sequences known to direct their passenger proteins into the SecB, signal recognition particle (SRP), or Twin-arginine translocation (Tat) pathway. In addition, AldO was fused to an ice nucleation protein (INP)-based anchoring motif for surface display. The results show that Tat-exported AldO and INP-surface-displayed AldO are active. The Tat-based system was successfully employed in converting xylitol by whole cells, whereas the use of the INP-based system was most likely restricted by lipopolysaccharide LPS in wild-type cells. It is anticipated that these whole-cell systems will be a valuable tool for further biological and industrial exploitation of AldO and other cofactor-containing enzymes.

Enzymatic synthesis of enantiopure alpha- and beta-amino acids by phenylalanine aminomutase-catalysed amination of cinnamic acid derivatives.

The phenylalanine aminomutase (PAM) from Taxus chinensis catalyses the conversion of alpha-phenylalanine to beta-phenylalanine, an important step in the biosynthesis of the N-benzoyl phenylisoserinoyl side-chain of the anticancer drug taxol. Mechanistic studies on PAM have suggested that (E)-cinnamic acid is an intermediate in the mutase reaction and that it can be released from the enzyme's active site. Here we describe a novel synthetic strategy that is based on the finding that ring-substituted (E)-cinnamic acids can serve as a substrate in PAM-catalysed ammonia addition reactions for the biocatalytic production of several important beta-amino acids. The enzyme has a broad substrate range and a high enantioselectivity with cinnamic acid derivatives; this allows the synthesis of several non-natural aromatic alpha- and beta-amino acids in excellent enantiomeric excess (ee >99 %). The internal 5-methylene-3,5-dihydroimidazol-4-one (MIO) cofactor is essential for the PAM-catalysed amination reactions. The regioselectivity of amination reactions was influenced by the nature of the ring substituent.

ADP competes with FAD binding in putrescine oxidase.

Putrescine oxidase from Rhodococcus erythropolis NCIMB 11540 (PuO(Rh)) is a soluble homodimeric flavoprotein of 100 kDa, which catalyzes the oxidative deamination of putrescine and some other aliphatic amines. The initial characterization of PuO(Rh) uncovered an intriguing feature: the enzyme appeared to contain only one noncovalently bound FAD cofactor per dimer. Here we show that this low FAD/protein ratio is the result of tight binding of ADP, thereby competing with FAD binding. MS analysis revealed that the enzyme is isolated as a mixture of dimers containing two molecules of FAD, two molecules ADP, or one FAD and one ADP molecule. In addition, based on a structural model of PuO(Rh) that was built using the crystal structure of human monoamine oxidase B (MAO-B), we constructed an active mutant enzyme, PuO(Rh) A394C, that contains covalently bound FAD. These findings show that the covalent FAD-protein linkage can be formed autocatalytically and hint to a new-found rationale for covalent flavinylation: covalent flavinylation may have evolved to prevent binding of ADP or related cellular compounds, which would prohibit formation of flavinylated and functional enzyme.

Labrys portucalensis sp. nov., a fluorobenzene-degrading bacterium isolated from an industrially contaminated sediment in northern Portugal.

A detailed classification of a novel bacterial strain, designated F11(T), capable of degrading fluorobenzene as a sole carbon and energy source, was performed by using a polyphasic approach. This Gram-negative, rod-shaped, non-motile, non-spore-forming, aerobic bacterium was isolated from a sediment sample collected from an industrially contaminated site in northern Portugal. The predominant whole-cell fatty acids were C(19 : 0) cyclo omega8c, C(16 : 0), C(18 : 1)omega7c, C(18 : 0), C(18 : 0) 3-OH and C(16 : 0) 3-OH. The G+C content of the DNA was 62.9 mol% and the major respiratory quinone was ubiquinone 10 (UQ-10). 16S rRNA gene sequence analysis revealed that strain F11(T) was a member of the class Alphaproteobacteria and was phylogenetically related to the genus Labrys, having sequence similarities of 95.6 and 93.1 % to the type strains of Labrys monachus and Labrys methylaminiphilus, respectively. DNA-DNA hybridization experiments revealed levels of relatedness of <70 % between strain F11(T) and the type strains of L. monachus and L. methylaminiphilus (38.6 and 34.1 %, respectively), justifying the classification of strain F11(T) as representing a novel species of the genus Labrys. The name Labrys portucalensis sp. nov. is proposed for this organism. The type strain is F11(T) (=LMG 23412(T)=DSM 17916(T)).

Identification of the catalytic residues of alpha-amino acid ester hydrolase from Acetobacter turbidans by labeling and site-directed mutagenesis.

The alpha-amino acid ester hydrolase from Acetobacter turbidans ATCC 9325 is capable of hydrolyzing and synthesizing the side chain peptide bond in beta-lactam antibiotics. Data base searches revealed that the enzyme contains an active site serine consensus sequence Gly-X-Ser-Tyr-X-Gly that is also found in X-prolyl dipeptidyl aminopeptidase. The serine hydrolase inhibitor p-nitrophenyl-p'-guanidino-benzoate appeared to be an active site titrant and was used to label the alpha-amino acid ester hydrolase. Electrospray mass spectrometry and tandem mass spectrometry analysis of peptides from a CNBr digest of the labeled protein showed that Ser(205), situated in the consensus sequence, becomes covalently modified by reaction with the inhibitor. Extended sequence analysis showed alignment of this Ser(205) with the catalytic nucleophile of some alpha/beta-hydrolase fold enzymes, which posses a catalytic triad composed of a nucleophile, an acid, and a base. Based on the alignments, 10 amino acids were selected for site-directed mutagenesis (Arg(85), Asp(86), Tyr(143), Ser(156), Ser(205), Tyr(206), Asp(338), His(370), Asp(509), and His(610)). Mutation of Ser(205), Asp(338,) or His(370) to an alanine almost fully inactivated the enzyme, whereas mutation of the other residues did not seriously affect the enzyme activity. Circular dichroism measurements showed that the inactivation was not caused by drastic changes in the tertiary structure. Therefore, we conclude that the catalytic domain of the alpha-amino acid ester hydrolase has an alpha/beta-hydrolase fold structure with a catalytic triad of Ser(205), Asp(338), and His(370). This distinguishes the alpha-amino acid ester hydrolase from the Ntn-hydrolase family of beta-lactam antibiotic acylases.

Molecular dynamics simulations as a tool for improving protein stability.

Haloalkane dehalogenase (DhlA) was used as a model protein to explore the possibility to use molecular dynamics (MD) simulations as a tool to identify flexible regions in proteins that can serve as a target for stability enhancement by introduction of a disulfide bond. DhlA consists of two domains: an alpha/beta-hydrolase fold main domain and a cap domain composed of five alpha-helices. MD simulations of DhlA showed high mobility in a helix-loop-helix region in the cap domain, involving residues 184-211. A disulfide cross-link was engineered between residue 201 of this flexible region and residue 16 of the main domain. The mutant enzyme showed substantial changes in both thermal and urea denaturation. The oxidized form of the mutant enzyme showed an increase of the apparent transition temperature from 47.5 to 52.5 degrees C, whereas the T(m,app) of the reduced mutant decreased by more than 8 degrees C compared to the wild-type enzyme. Urea denaturation results showed a similar trend. Measurement of the kinetic stability showed that the introduction of the disulfide bond caused a decrease in activation free energy of unfolding of 0.43 kcal mol(-1) compared to the wild-type enzyme and also indicated that the helix-loop-helix region was involved early in the unfolding process. The results show that MD simulations are capable of identifying mobile protein domains that can successfully be used as a target for stability enhancement by the introduction of a disulfide cross-link.

Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding an alpha-amino acid ester hydrolase from Acetobacter turbidans.

The alpha-amino acid ester hydrolase from Acetobacter turbidans ATCC 9325 is capable of hydrolyzing and synthesizing beta-lactam antibiotics, such as cephalexin and ampicillin. N-terminal amino acid sequencing of the purified alpha-amino acid ester hydrolase allowed cloning and genetic characterization of the corresponding gene from an A. turbidans genomic library. The gene, designated aehA, encodes a polypeptide with a molecular weight of 72,000. Comparison of the determined N-terminal sequence and the deduced amino acid sequence indicated the presence of an N-terminal leader sequence of 40 amino acids. The aehA gene was subcloned in the pET9 expression plasmid and expressed in Escherichia coli. The recombinant protein was purified and found to be dimeric with subunits of 70 kDa. A sequence similarity search revealed 26% identity with a glutaryl 7-ACA acylase precursor from Bacillus laterosporus, but no homology was found with other known penicillin or cephalosporin acylases. There was some similarity to serine proteases, including the conservation of the active site motif, GXSYXG. Together with database searches, this suggested that the alpha-amino acid ester hydrolase is a beta-lactam antibiotic acylase that belongs to a class of hydrolases that is different from the Ntn hydrolase superfamily to which the well-characterized penicillin acylase from E. coli belongs. The alpha-amino acid ester hydrolase of A. turbidans represents a subclass of this new class of beta-lactam antibiotic acylases.