Juergen Pleiss

The Cytochrome P450 Engineering Database

SUMMARY The Cytochrome P450 Engineering Database (CYPED) has been designed to serve as a tool for a comprehensive and systematic comparison of protein sequences and structures within the vast and diverse family of cytochrome P450 monooxygenases (CYPs). The CYPED currently integrates sequence and structure data of 3911 and 25 proteins, respectively. Proteins are grouped into homologous families and superfamilies according to Nelsons classification. Nonclassified CYP sequences are assigned by similarity. Functionally relevant residues are annotated. The web accessible version contains multisequence alignments, phylogenetic trees and HMM profiles. The CYPED is regularly updated and supplies all data for download. Thus, it provides a valuable data source for phylogenetic analysis, investigation of sequence-function relationships and the design of CYPs with improved biochemical properties. ABBREVIATIONS Cytochrome P450 Engineering Database, CYPED; cytochrome P450 monooxygenase, CYP; Hidden Markov Model, HMM. AVAILABILITY www.cyped.uni-stuttgart.de

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.

Engineering of Candida antarctica lipase B for hydrolysis of bulky carboxylic acid esters.

Candida antarctica lipase B (CALB) is a widely used biocatalyst with high activity and specificity for a wide range of primary and secondary alcohols. However, the range of converted carboxylic acids is more narrow and mainly limited to unbranched fatty acids. To further broaden the biotechnological applications of CALB it is of interest to expand the range of converted carboxylic acid and extend it to carboxylic acids that are branched or substituted in close proximity of the carboxyl group. An in silico library of 2400 CALB variants was built and screened in silico by substrate-imprinted docking, a four step docking procedure. First, reaction intermediates of putative substrates are covalently docked into enzyme active sites. Second, the geometry of the resulting enzyme-substrate complex is optimized. Third, the substrate is removed from the complex and then docked again into the optimized structure. Fourth, the resulting substrate poses are rated by geometric filter criteria as productive or non-productive poses. Eleven enzyme variants resulting from the in silico screening were expressed in Escherichia coli BL21 and measured in the hydrolysis of two branched fatty acid esters, isononanoic acid ethyl ester and 2-ethyl hexanoic acid ethyl esters. Five variants showed an initial increase in activity. The variant with the highest wet mass activity (T138S) was purified and further characterized. It showed a 5-fold increase in hydrolysis of isononanoic acid ethyl ester, but not toward sterically more demanding 2-ethyl hexanoic acid ethyl ester.

Structural basis of steroid binding and oxidation by the cytochrome P450 CYP109E1 from Bacillus megaterium.

Cytochrome P450 monooxygenases (P450s) are attractive enzymes for the pharmaceutical industry, in particular, for applications in steroidal drug synthesis. Here, we report a comprehensive functional and structural characterization of CYP109E1, a novel steroid‐converting cytochrome P450 enzyme identified from the genome of Bacillus megaterium DSM319. In vitro and whole‐cell in vivo turnover experiments, combined with binding assays, revealed that CYP109E1 is able to hydroxylate testosterone at position 16β. Related steroids with bulky substituents at carbon C17, like corticosterone, bind to the enzyme without being converted. High‐resolution X‐ray structures were solved of a steroid‐free form of CYP109E1 and of complexes with testosterone and corticosterone. The structural analysis revealed a highly dynamic active site at the distal side of the heme, which is wide open in the absence of steroids, can bind four ordered corticosterone molecules simultaneously, and undergoes substantial narrowing upon binding of single steroid molecules. In the crystal structures, the single bound steroids adopt unproductive binding modes coordinating the heme‐iron with their C3‐keto oxygen. Molecular dynamics (MD) simulations suggest that the steroids may also bind in ~180° reversed orientations with the C16 carbon and C17‐substituents pointing toward the heme, leading to productive binding of testosterone explaining the observed regio‐ and stereoselectivity. The X‐ray structures and MD simulations further identify several residues with important roles in steroid binding and conversion, which could be confirmed by site‐directed mutagenesis. Taken together, our results provide unique insights into the CYP109E1 activity, substrate specificity, and regio/stereoselectivity.

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

BackgroundSHV 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 (http://www.lahey.org/Studies/). 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.DescriptionThe 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.The SHVED is accessible at http://www.LacED.uni-stuttgart.de/classA/SHVED/. 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.ConclusionsThe 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.

Morphing Activity between Structurally Similar Enzymes: From Heme-Free Bromoperoxidase to Lipase

In this study, to explore the plasticity of the alpha/beta-hydrolase fold family, we converted bromoperoxidase A2 (BPO-A2) from Streptomyces aureofaciens to a lipase by structure comparison with lipase A (LipA) from Bacillus subtilis. These two enzymes have similar structures (2.1 A rmsd) and a very low level of sequence identity ( approximately 18%). A variant BL1 was constructed by deleting the caplike domain of BPO-A2 and further fine-tuning the newly formed substrate binding site. The lipase activity was successfully transplanted on BL1, while the halogenation activity was totally lost. BL1 also showed higher hydrolytic activities toward long chain p-nitrophenyl esters, such as p-nitrophenyl caprylate (3.7-fold) and p-nitrophenyl palmitate (7.0-fold), while its activity toward a short chain ester (p-nitrophenyl acetate) decreased dramatically, to only 1.2% of that of BPO-A2. After two rounds of directed evolution and site-directed mutagenesis on selected residues, several mutants with both improved hydrolytic activities and substrate preferences toward long chain substrates were obtained. The highest hydrolytic activity toward p-nitrophenyl palmitate of the best mutant BL1-2-E8-plusI was improved by 40-fold compared with that of BL1. These results demonstrate the possibility of manipulating the caplike domain of alpha/beta-hydrolase fold enzymes and provide further understanding of the structure-function relationship of the alpha/beta-hydrolase fold enzymes. The design strategy used in this study could serve as a useful approach for constructing variants with targeted catalytic properties using the alpha/beta-hydrolase fold.

Reply to “The Curious Case of TEM-116”

In their comment letter “The curious case of TEM-116” (1), Jacoby and Bush discuss the long-standing question of whether the TEM -lactamase A184V V84I variant (TEM-116) is a natural variant. The fact that TEM-116 has a high centrality in the sequence similarity network of TEM -lactamase variants (2) indicates that “TEM-116 is now a naturally occurring enzyme” (1). TEM-116 has been identified multiple times in clinical isolates since 2004 (3). However, in 2007, it was suspected that the detection of TEM-116 might just be a false-positive result, caused by a contamination in PCR reagents (4), since the TEM -lactamase A184V V84I double mutant has been part of the plasmid pUC5 and its plasmid progeny since 1982 (5). Therefore, the question arose as to whether TEM-116 is a product of evolution or merely an engineered enzyme. It would not have been the first time that naturally occurring TEM variants were first developed synthetically and detected in clinical isolates only later; an example is the E104K M182T G238S triple variant, which was developed by DNA shuffling in 1994 (6) and detected in a clinical isolate in 1998 (7). In the meantime, a large number of functional TEM -lactamase variants were identified by directed-evolution experiments, and more than 200 naturally occurring TEM -lactamase variants are listed in the TEM mutation table maintained at the Lahey Clinic (8). These data show that stabilization, ESBL activity, or inhibitor resistance is mediated by mutations at fewer than 15 of the most relevant hot spot positions. In the literature as well as in the comment letter by Jacoby and Bush (1), a sharp distinction is made between natural and synthetic TEM -lactamase variants. However, we expect that a large fraction of (if not all) functional variants are expressed by some of the 10 cells in the contemporary biosphere (9), since genes conferring resistance to -lactams existed long before the advent of antibiotics in clinical use 70 years ago, as shown by metagenomic analyses of 30,000-year-old permafrost sediments (10). While it is still unknown how many of the 20 ( 3 · 10) possible variants resulting from variations at 15 hot spot positions are functional, they may easily have been screened by the 10 cells that came into life during 4 gigayears of evolution (9), even if only a small fraction of them expressed a TEM -lactamase variant. Thus, we expect that each functional TEM -lactamase variant that we find by mutating the hot spot positions is also a member of the large pool of natural variants. Our current knowledge of the network of functional TEM -lactamase variants is still sparse (2), but we are gradually detecting new functional variants, either by sequencing of clinical isolates or by synthetic approaches, such as directed evolution. The scientific challenge is to understand (and predict) which of the 3 · 10 possible variants are functional.