Dooil Kim

Biosynthesis of the Allylmalonyl-CoA Extender Unit for the FK506 Polyketide Synthase Proceeds through a Dedicated Polyketide Synthase and Facilitates the Mutasynthesis of Analogues

The allyl moiety of the immunosuppressive agent FK506 is structurally unique among polyketides and critical for its potent biological activity. Here, we detail the biosynthetic pathway to allylmalonyl-coenzyme A (CoA), from which the FK506 allyl group is derived, based on a comprehensive chemical, biochemical, and genetic interrogation of three FK506 gene clusters. A discrete polyketide synthase (PKS) with noncanonical domain architecture presumably in coordination with the fatty acid synthase pathway of the host catalyzes a multistep enzymatic reaction to allylmalonyl-CoA via trans-2-pentenyl-acyl carrier protein. Characterization of this discrete pathway facilitated the engineered biosynthesis of novel allyl group-modified FK506 analogues, 36-fluoro-FK520 and 36-methyl-FK506, the latter of which exhibits improved neurite outgrowth activity. This unique feature of FK506 biosynthesis, in which a dedicated PKS provides an atypical extender unit for the main modular PKS, illuminates a new strategy for the combinatorial biosynthesis of designer macrolide scaffolds as well as FK506 analogues.

The acetylproteome of Gram-positive model bacterium Bacillus subtilis

Nε‐lysine acetylation, a reversible and highly regulated PTM, has been shown to occur in the model Gram‐negative bacteria Escherichia coli and Salmonella enterica. Here, we extend this acetylproteome analysis to Bacillus subtilis, a model Gram‐positive bacterium. Through anti‐acetyllysine antibody‐based immunoseparation of acetylpeptides followed by nano‐HPLC/MS/MS analysis, we identified 332 unique lysine‐acetylated sites on 185 proteins. These proteins are mainly involved in cellular housekeeping functions such as central metabolism and protein synthesis. Fifity‐nine of the lysine‐acetylated proteins showed homology with lysine‐acetylated proteins previously identified in E. coli, suggesting that acetylated proteins are more conserved. Notably, acetylation was found at or near the active sites predicted by Prosite signature, including SdhA, RocA, Kbl, YwjH, and YfmT, indicating that lysine acetylation may affect their activities. In 2‐amino‐3‐ketobutyrate CoA ligase Kbl, a class II aminotransferase, a lysine residue involved in pyridoxal phosphate attachment was found to be acetylated. This data set provides evidence for the generality of lysine acetylation in eubacteria and opens opportunities to explore the consequences of acetylation modification on the molecular physiology of B. subtilis.

Proteomic analysis of acetylation in thermophilic Geobacillus kaustophilus

Recent analysis of prokaryotic Nε‐lysine‐acetylated proteins highlights the posttranslational regulation of a broad spectrum of cellular proteins. However, the exact role of acetylation remains unclear due to a lack of acetylated proteome data in prokaryotes. Here, we present the Nε‐lysine‐acetylated proteome of gram‐positive thermophilic Geobacillus kaustophilus. Affinity enrichment using acetyl‐lysine‐specific antibodies followed by LC‐MS/MS analysis revealed 253 acetylated peptides representing 114 proteins. These acetylated proteins include not only common orthologs from mesophilic Bacillus counterparts, but also unique G. kaustophilus proteins, indicating that lysine acetylation is pronounced in thermophilic bacteria. These data complement current knowledge of the bacterial acetylproteome and provide an expanded platform for better understanding of the function of acetylation in cellular metabolism.

Discovery of parallel pathways of kanamycin biosynthesis allows antibiotic manipulation

Kanamycin is one of the most widely used antibiotics, yet its biosynthetic pathway remains unclear. Current proposals suggest that the kanamycin biosynthetic products are linearly related via single enzymatic transformations. To explore this system, we have reconstructed the entire biosynthetic pathway through the heterologous expression of combinations of putative biosynthetic genes from Streptomyces kanamyceticus in the non-aminoglycoside-producing Streptomyces venezuelae. Unexpectedly, we discovered that the biosynthetic pathway contains an early branch point, governed by the substrate promiscuity of a glycosyltransferase, that leads to the formation of two parallel pathways in which early intermediates are further modified. Glycosyltransferase exchange can alter flux through these two parallel pathways, and the addition of other biosynthetic enzymes can be used to synthesize known and new highly active antibiotics. These results complete our understanding of kanamycin biosynthesis and demonstrate the potential of pathway engineering for direct in vivo production of clinically useful antibiotics and more robust aminoglycosides.

Engineering Bacterial Cytochrome P450 (P450) BM3 into a Prototype with Human P450 Enzyme Activity Using Indigo Formation

Human cytochrome P450 (P450) enzymes metabolize a variety of endogenous and xenobiotic compounds, including steroids, drugs, and environmental chemicals. In this study, we examine the possibility that bacterial P450 BM3 (CYP102A1) mutants with indole oxidation activity have the catalytic activities of human P450 enzymes. Error-prone polymerase chain reaction was carried out on the heme domain-coding region of the wild-type gene to generate a CYP102A1 DNA library. The library was transformed into Escherichia coli for expression of the P450 mutants. A colorimetric colony-based method was adopted for primary screening of the mutants. When the P450 activities were measured at the whole-cell level, some of the blue colonies, but not the white colonies, possessed apparent oxidation activity toward coumarin and 7-ethoxycoumarin, which are typical human P450 substrates that produce fluorescent products. Coumarin is oxidized by the CYP102A1 mutants to produce two metabolites, 7-hydroxycoumarin and 3-hydroxycoumarin. In addition, 7-ethoxycoumarin is simultaneously oxidized to 7-hydroxycoumarin by O-deethylation reaction and to 3-hydroxy,7-ethoxycoumarin by 3-hydroxylation reactions. Highly active mutants are also able to metabolize several other human P450 substrates, including phenacetin, ethoxyresorufin, and chlorzoxazone. These results indicate that indigo formation provides a simple assay for identifying CYP102A1 mutants with a greater potential for human P450 activity. Furthermore, our computational findings suggest a correlation between the stabilization of the binding site and the catalytic efficiency of CYP102A1 mutants toward coumarin: the more stable the structure in the binding site, the lower the energy barrier and the higher the catalytic efficiency.

Application of rpoB and Zinc Protease Gene for Use in Molecular Discrimination of Fusobacterium nucleatum Subspecies

ABSTRACT Fusobacterium nucleatum is classified into five subspecies that inhabit the human oral cavity (F. nucleatum subsp. nucleatum, F. nucleatum subsp. polymorphum, F. nucleatum subsp. fusiforme, F. nucleatum subsp. vincentii, and F. nucleatum subsp. animalis) based on several phenotypic characteristics and DNA-DNA hybridization patterns. However, the methods for detecting or discriminating the clinical isolates of F. nucleatum at the subspecies levels are laborious, expensive, and time-consuming. Therefore, in this study, the nucleotide sequences of the RNA polymerase β-subunit gene (rpoB) and zinc protease gene were analyzed to discriminate the subspecies of F. nucleatum. The partial sequences of rpoB (approximately 2,419 bp), the zinc protease gene (878 bp), and 16S rRNA genes (approximately 1,500 bp) of the type strains of five subspecies, 28 clinical isolates of F. nucleatum, and 10 strains of F. periodonticum (as a control group) were determined and analyzed. The phylogenetic data showed that the rpoB and zinc protease gene sequences clearly delineated the subspecies of F. nucleatum and provided higher resolution than the 16S rRNA gene sequences in this respect. According to the phylogenetic analysis of rpoB and the zinc protease gene, F. nucleatum subsp. vincentii and F. nucleatum subsp. fusiforme might be classified into a single subspecies. Five clinical isolates could be delineated as a new subspecies of F. nucleatum. The results suggest that rpoB and the zinc protease gene are efficient targets for the discrimination and taxonomic analysis of the subspecies of F. nucleatum.

GENERATION OF HUMAN CHIRAL METABOLITES OF SIMVASTATIN AND LOVASTATIN BY BACTERIAL CYP102A1 MUTANTS

Recently, the wild-type and mutant forms of cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium were found to oxidize various xenobiotic substrates, including pharmaceuticals, of human P450 enzymes. Simvastatin and lovastatin, which are used to treat hyperlipidemia and hypercholesterolemia, are oxidized by human CYP3A4/5 to produce several metabolites, including 6′β-hydroxy (OH), 3″-OH, and exomethylene products. In this report, we show that the oxidation of simvastatin and lovastatin was catalyzed by wild-type CYP102A1 and a set of its mutants, which were generated by site-directed and random mutagenesis. One major hydroxylated product (6′β-OH) and one minor product (6′-exomethylene), but not other products, were produced by CYP102A1 mutants. Formation of the metabolites was confirmed by high-performance liquid chromatography, liquid chromatography-mass spectroscopy, and NMR. Chemical methods to synthesize the metabolites of simvastatin and lovastatin have not been reported. These results demonstrate that CYP102A1 mutants can be used to produce human metabolites, especially chiral metabolites, of simvastatin and lovastatin. Our computational findings suggest that a conformational change in the cavity of the mutant active sites is related to the activity change. The modeling results also suggest that the activity change results from the movement of several specific residues in the active sites of the mutants. Furthermore, our computational findings suggest a correlation between the stabilization of the binding site and the catalytic efficiency of CYP102A1 mutants toward simvastatin and lovastatin.

Production of high-content inulo-oligosaccharides from inulin by a purified endoinulinase

Inulo-oligosaccharides were produced from inulin with high yield by using a purified endoinulinase from a commercial inulinase preparation. The maximum yield of oligosaccharide achieved was around 96% irrespective of substrate concentrations ranged from 50 to 150 g inulin/l. A wide range of degradation products from inulin, varying in their DP (degree of polymerization) 2 to 6, were obtained where the major oligosaccharides were DP3 and 4. The reaction pH gave rise to a significant difference in yield and sugar composition of inulo-oligosaccharides.

Crystal structure of the apo form of D‐alanine: D‐alanine ligase (Ddl) from Thermus caldophilus: A basis for the substrate‐induced conformational changes

Introduction. D-alanine:D-alanine ligase (Ddl) catalyses the dimerization of D-alanine before its incorporation in peptidoglycan precursors. The synthesis of D-alanine:Dalanine begins with an attack on the first D-alanine by the -phosphate of adenosine triphosphate (ATP) to yield an acylphosphate. That is followed by attack by the amino group of the second D-alanine, which eliminates the phosphate and produces the D-alanine: D-alanine dipeptide. Peptidoglycan biosynthesis has long been an attractive target for antibacterial drugs, such as D-cycloserine, glycopeptide antibiotics (vancomycin and teicoplanins), and -lactams (penicillin and cephalosporins). Vancomycintype antibiotics, for example, bind directly to the D-alanine: D-alanine terminus, thereby inhibiting crosslinking by the transpeptidase. Notably, bacteria that show vancomycin resistance, which develops after prolonged clinical treatment with vancomycin, possess an inactive Ddl and rely on another ligase, D-alanine:D-lactate ligase (Van), which produces D-alanine:D-lactate rather than D-alanine:Dalanine for cell wall synthesis. The switch from D-alanine: D-alanine peptidoglycan termini to D-alanine:D-lactate results in the loss of crucial hydrogen bonding interactions that causes a 1000-fold reduction in vancomycin binding affinity. X-ray crystallographic studies of Ddl and Van have contributed significantly to our understanding of the ligand specificity these two enzymes and suggest that a His residue in Van plays a critical role. A positive charge on the side chain imidazole nitrogen of His would attract the negatively charged lactate to the second substrate binding site at pH values less than 7, but at higher pH values Van would predominantly synthesize D-alanine:D-alanine. In Ddl, a Tyr residue [Tyr216 in Escherichia coli (Eco) DdlB, Tyr232 in Thermus caldophilus (Tca) Ddl] occupies the same spatial position as the His residue, and the hydroxyl group of the Tyr interacts with the COOH-terminal of the second D-alanine substrate. The structure of Eco DdlB complexed with ADP/ phosphorylated phosphinate (PDB ID: 2DLN) or with ADP/phosphorylated phosphonate (PDB ID: 1IOV) has been determined, as have the structures of Leuconostoc mesenteroides (Lme) D-Alanine:D-Lactate ligase complexed with ADP and phosphinophosphate (PDB ID:1EHI) and Enterococcus faecium (Efa) VanA complexed with ADP and phosphinophosphate (PDB ID:1E4E). However, to analyze the reaction mechanisms of these enzymes and their associated conformational changes, it is necessary to know the structures of both the substrate-bound and substrate-free forms of these enzymes. Our aim in the present study, therefore, was to grow crystals of Ddl that diffracted to high resolution in the absence of substrates. Here we report the X-ray structure of TcaDdl resolved to a resolution of 1.9 A and describe the conformational differences of the apo structure, comparing it with the structures of the previously described transition state analogue complex.

Asymmetric synthesis of (S)-ethyl-4-chloro-3-hydroxy butanoate using a Saccharomyces cerevisiae reductase: Enantioselectivity and enzyme–substrate docking studies

Ethyl (S)-4-chloro-3-hydroxy butanoate (ECHB) is a building block for the synthesis of hypercholesterolemia drugs. In this study, various microbial reductases have been cloned and expressed in Escherichia coli. Their reductase activities toward ethyl-4-chloro oxobutanoate (ECOB) have been assayed. Amidst them, Bakers yeast YDL124W, YOR120W, and YOL151W reductases showed high activities. YDL124W produced (S)-ECHB exclusively, whereas YOR120W and YOL151W made (R)-form alcohol. The homology models and docking models with ECOB and NADPH elucidated their substrate specificities and enantioselectivities. A glucose dehydrogenase-coupling reaction was used as NADPH recycling system to perform continuously the reduction reaction. Recombinant E. coli cell co-expressing YDL124W and Bacillus subtilis glucose dehydrogenase produced (S)-ECHB exclusively.