Structure and dynamics of Candida rugosa lipase: the role of organic solvent

The effect of organic solvent on the structure and dynamics of proteins was investigated by multiple molecular dynamics simulations (1 ns each) of Candida rugosa lipase in water and in carbon tetrachloride. The choice of solvent had only a minor structural effect. For both solvents the open and the closed conformation of the lipase were near to their experimental X-ray structures (Cα rms deviation 1-1.3 Å). However, the solvents had a highly specific effect on the flexibility of solvent-exposed side chains: polar side chains were more flexible in water, but less flexible in organic solvent. In contrast, hydrophobic residues were more flexible in organic solvent, but less flexible in water. As a major effect solvent changed the dynamics of the lid, a mobile element involved in activation of the lipase, which fluctuated as a rigid body about its average position. While in water the deviations were about 1.6 Å, organic solvent reduced flexibility to 0.9 Å. This increase rigidity was caused by two salt bridges (Lys85-Asp284, Lys75-Asp79) and a stable hydrogen bond (Lys75-Asn 292) in organic solvent. Thus, organic solvents stabilize the lid but render the side chains in the hydrophobic substrate-binding site more mobile. © Springer-Verlag 2004

Inverting the stereoselectivity of an NADH-dependent imine reductase variant

Imine reductases (IREDs) offer biocatalytic routes to chiral amines and have a natural preference for the NADPH cofactor. In previous work, we reported enzyme engineering of the (R)-selective IRED from Myxococcus stipitatus (NADH-IRED-Ms) yielding a NADH-dependent variant with high catalytic efficiency. However, no IRED with NADH specificity and (S)-selectivity in asymmetric reductions has yet been reported. Herein, we applied semi-rational enzyme engineering to switch the selectivity of NADH-IRED-Ms. The quintuple variant A241V/H242Y/N243D/V244Y/A245L showed reverse stereopreference in the reduction of the cyclic imine 2- methylpyrroline compared to the wild-type and afforded the (S)- amine product with >99% conversion and 91% enantiomeric excess. We also report the crystal-structures of the NADPH-dependent (R)- IRED-Ms wild-type enzyme and the NADH-dependent NADH-IREDMs variant and molecular dynamics (MD) simulations to rationalize the inverted stereoselectivity of the quintuple variant

The Bacteroidetes Aequorivita sp. and Kaistella jeonii Produce Promiscuous Esterases With PET-Hydrolyzing Activity

Certain members of the Actinobacteria and Proteobacteria are known to degrade polyethylene terephthalate (PET). Here, we describe the first functional PET-active enzymes from the Bacteroidetes phylum. Using a PETase-specific Hidden-Markov-Model- (HMM-) based search algorithm, we identified several PETase candidates from Flavobacteriaceae and Porphyromonadaceae. Among them, two promiscuous and cold-active esterases derived from Aequorivita sp. (PET27) and Kaistella jeonii (PET30) showed depolymerizing activity on polycaprolactone (PCL), amorphous PET foil and on the polyester polyurethane Impranil® DLN. PET27 is a 37.8 kDa enzyme that released an average of 174.4 nmol terephthalic acid (TPA) after 120 h at 30°C from a 7 mg PET foil platelet in a 200 μl reaction volume, 38-times more than PET30 (37.4 kDa) released under the same conditions. The crystal structure of PET30 without its C-terminal Por-domain (PET30ΔPorC) was solved at 2.1 Å and displays high structural similarity to the IsPETase. PET30 shows a Phe-Met-Tyr substrate binding motif, which seems to be a unique feature, as IsPETase, LCC and PET2 all contain Tyr-Met-Trp binding residues, while PET27 possesses a Phe-Met-Trp motif that is identical to Cut190. Microscopic analyses showed that K. jeonii cells are indeed able to bind on and colonize PET surfaces after a few days of incubation. Homologs of PET27 and PET30 were detected in metagenomes, predominantly aquatic habitats, encompassing a wide range of different global climate zones and suggesting a hitherto unknown influence of this bacterial phylum on man-made polymer degradation

SHV Lactamase Engineering Database: a reconciliation tool for SHV β-lactamases in public databases

<p>Abstract</p> <p>Background</p> <p>SHV β-lactamases confer resistance to a broad range of antibiotics by accumulating mutations. The number of SHV variants is steadily increasing. 117 SHV variants have been assigned in the SHV mutation table (<url>http://www.lahey.org/Studies/</url>). Besides, information about SHV β-lactamases can be found in the rapidly growing NCBI protein database. The SHV β-Lactamase Engineering Database (SHVED) has been developed to collect the SHV β-lactamase sequences from the NCBI protein database and the SHV mutation table. It serves as a tool for the detection and reconciliation of inconsistencies, and for the identification of new SHV variants and amino acid substitutions.</p> <p>Description</p> <p>The SHVED contains 200 protein entries with distinct sequences and 20 crystal structures. 83 protein sequences are included in the both the SHV mutation table and the NCBI protein database, while 35 and 82 protein sequences are only in the SHV mutation table and the NCBI protein database, respectively. Of these 82 sequences, 41 originate from microbial sources, and 22 of them are full-length sequences that harbour a mutation profile which has not been classified yet in the SHV mutation table. 27 protein entries from the NCBI protein database were found to have an inconsistency in SHV name identification. These inconsistencies were reconciled using information from the SHV mutation table and stored in the SHVED.</p> <p>The SHVED is accessible at <url>http://www.LacED.uni-stuttgart.de/classA/SHVED/</url>. It provides sequences, structures, and a multisequence alignment of SHV β-lactamases with the corrected annotation. Amino acid substitutions at each position are also provided. The SHVED is updated monthly and supplies all data for download.</p> <p>Conclusions</p> <p>The SHV β-Lactamase Engineering Database (SHVED) contains information about SHV variants with reconciled annotation. It serves as a tool for detection of inconsistencies in the NCBI protein database, helps to identify new mutations resulting in new SHV variants, and thus supports the investigation of sequence-function relationships of SHV β-lactamases.</p

Modeling Domino Effects in Enzymes: Molecular Basis of the Substrate Specificity of the Bacterial Metallo-β-lactamases IMP-1 and IMP-6

Metallo-β-lactamases can hydrolyze a broad spectrum of β-lactam antibiotics and thus confer resistance to bacteria. For the Pseudomonas aeruginosa enzyme IMP-1, several variants have been reported. IMP-6 and IMP-1 differ by a single residue (glycine and serine at position 196, respectively), but have significantly different substrate spectra; while the catalytic efficiency toward the two cephalosporins cephalothin and cefotaxime is similar for both variants, IMP-1 is up to 10-fold more efficient than IMP-6 toward cephaloridine and ceftazidime. Interestingly, this biochemical effect is caused by a residue remote from the active site. The substrate-specific impact of residue 196 was studied by molecular dynamics simulations using a cationic dummy atom approach for the zinc ions. Substrates were docked in an intermediate structure near the transition state to the binding site of IMP-1 and IMP-6. At a simulation temperature of 100 K, most complexes were stable during 1 ns of simulation time. However, at higher temperatures, some complexes became unstable and the substrate changed to a nonactive conformation. To model stability, six molecular dynamics simulations at 100 K were carried out for all enzyme−substrate complexes. Stable structures were further heated to 200 and 300 K. By counting stable structures, we derived a stability ranking score which correlated with experimentally determined catalytic efficiency. The use of a stability score as an indicator of catalytic efficiency of metalloenzymes is novel, and the study of substrates in a near-transition state intermediate structure is superior to the modeling of Michaelis complexes. The remote effect of residue 196 can be described by a domino effect:  upon replacement of serine with glycine, a hole is created and a stabilizing interaction between Ser196 and Lys33 disappears, rendering the neighboring residues more flexible; this increased flexibility is then transferred to the active site

Impact of remote mutations on metallo-β-lactamase substrate specificity: implications for the evolution of antibiotic resistance

Metallo-β-lactamases have raised concerns due to their ability to hydrolyze a broad spectrum of β-lactam antibiotics. The G262S point mutation distinguishing the metallo-β-lactamase IMP-1 from IMP-6 has no effect on the hydrolysis of the drugs cephalothin and cefotaxime, but significantly improves catalytic efficiency toward cephaloridine, ceftazidime, benzylpenicillin, ampicillin, and imipenem. This change in specificity occurs even though residue 262 is remote from the active site. We investigated the substrate specificities of five other point mutants resulting from single-nucleotide substitutions at positions near residue 262: G262A, G262V, S121G, F218Y, and F218I. The results suggest two types of substrates: type I (nitrocefin, cephalothin, and cefotaxime), which are converted equally well by IMP-6, IMP-1, and G262A, but even more efficiently by the other mutants, and type II (ceftazidime, benzylpenicillin, ampicillin, and imipenem), which are hydrolyzed much less efficiently by all the mutants. G262V, S121G, F218Y, and F218I improve conversion of type I substrates, whereas G262A and IMP-1 improve conversion of type II substrates, indicating two distinct evolutionary adaptations from IMP-6. Substrate structure may explain the catalytic efficiencies observed. Type I substrates have R2 electron donors, which may stabilize the substrate intermediate in the binding pocket. In contrast, the absence of these stabilizing interactions with type II substrates may result in poor conversion. This observation may assist future drug design. As the G262A and F218Y mutants confer effective resistance to Escherichia coli BL21(DE3) cells (high minimal inhibitory concentrations), they are likely to evolve naturally

Modeling Domino Effects in Enzymes: Molecular Basis of the Substrate Specificity of the Bacterial Metallo-β-lactamases IMP-1 and IMP-6

Metallo-β-lactamases can hydrolyze a broad spectrum of β-lactam antibiotics and thus confer resistance to bacteria. For the Pseudomonas aeruginosa enzyme IMP-1, several variants have been reported. IMP-6 and IMP-1 differ by a single residue (glycine and serine at position 196, respectively), but have significantly different substrate spectra; while the catalytic efficiency toward the two cephalosporins cephalothin and cefotaxime is similar for both variants, IMP-1 is up to 10-fold more efficient than IMP-6 toward cephaloridine and ceftazidime. Interestingly, this biochemical effect is caused by a residue remote from the active site. The substrate-specific impact of residue 196 was studied by molecular dynamics simulations using a cationic dummy atom approach for the zinc ions. Substrates were docked in an intermediate structure near the transition state to the binding site of IMP-1 and IMP-6. At a simulation temperature of 100 K, most complexes were stable during 1 ns of simulation time. However, at higher temperatures, some complexes became unstable and the substrate changed to a nonactive conformation. To model stability, six molecular dynamics simulations at 100 K were carried out for all enzyme−substrate complexes. Stable structures were further heated to 200 and 300 K. By counting stable structures, we derived a stability ranking score which correlated with experimentally determined catalytic efficiency. The use of a stability score as an indicator of catalytic efficiency of metalloenzymes is novel, and the study of substrates in a near-transition state intermediate structure is superior to the modeling of Michaelis complexes. The remote effect of residue 196 can be described by a domino effect:  upon replacement of serine with glycine, a hole is created and a stabilizing interaction between Ser196 and Lys33 disappears, rendering the neighboring residues more flexible; this increased flexibility is then transferred to the active site