An Enzyme Containing the Conserved Domain of Unknown Function DUF62 Acts as a Stereoselective (Rs ,Sc )-S-Adenosylmethionine Hydrolase.

Homochirality is a signature of biological systems. The essential and ubiquitous cofactor S-adenosyl-l-methionine (SAM) is synthesized in cells from adenosine triphosphate and l-methionine to yield exclusively the (S,S)-SAM diastereomer. (S,S)-SAM plays a crucial role as the primary methyl donor in transmethylation reactions important to the development and homeostasis of all organisms from bacteria to humans. However, (S,S)-SAM slowly racemizes at the sulfonium center to yield the inactive (R,S)-SAM, which can inhibit methyltransferases. Control of SAM homochirality has been shown to involve homocysteine S-methyltransferases in plants, insects, worms, yeast, and in ∼18 % of bacteria. Herein, we show that a recombinant protein containing a domain of unknown function (DUF62) from the actinomycete bacterium Salinispora tropica functions as a stereoselective (R,S)-SAM hydrolase (adenosine-forming). DUF62 proteins are encoded in the genomes of 21 % of bacteria and 42 % of archaea and potentially represent a novel mechanism to remediate SAM damage.

Cloning, Expression and Characterization of a Novel α-Amylase from Salinispora arenicola CNP193.

α-Amylases are used in various biotechnological processes including the textile, paper, food, biofuels, detergents and pharmaceutical industries. In this study, a novel gene encoding α-amylase was cloned from marine bacterium Salinispora arenicola CNP193 and the protein was expressed in Escherichia coli. The α-amylase gene from S. arenicola CNP193 had a length of 1839 bp and encoded a α-amylase with an estimated molecular mass of 74 kDa. The optimum temperature and pH for the recombinant α-amylase was 50 °C and 7 respectively. Na+, K+ and Ca2+ increased the activity of the recombinant α-amylase whereas the enzyme was inhibited by Cu2+, Zn2+, Hg2+, Pb2+, Fe3+ and Mn2+. Thin layer chromatography results confirmed that monosaccharide, disaccharide and maltotriose are the hydrolysis products. The results of our study suggest that this enzyme has considerable potential in industrial applications.

Structural Basis of Tryptophan Reverse N-Prenylation Catalyzed by CymD.

Indole prenyltransferases catalyze the prenylation of l-tryptophan (l-Trp) and other indoles to produce a diverse set of natural products in bacteria, fungi, and plants, many of which possess useful biological properties. Among this family of enzymes, CymD from Salinispora arenicola catalyzes the reverse N1 prenylation of l-Trp, an unusual reaction given the poor nucleophilicity of the indole nitrogen. CymD utilizes dimethylallyl diphosphate (DMAPP) as the prenyl donor, catalyzing the dissociation of the diphosphate leaving group followed by nucleophilic attack of the indole nitrogen at the tertiary carbon of the dimethylallyl cation. To better understand the structural basis of selective indole N-alkylation reactions in biology, we have determined the X-ray crystal structures of CymD, the CymD-l-Trp complex, and the CymD-l-Trp-DMSPP complex (DMSPP is dimethylallyl S-thiolodiphosphate, an unreactive analogue of DMAPP). The orientation of l-Trp with respect to DMSPP reveals how the active site contour of CymD serves as a template to direct the reverse prenylation of the indole nitrogen. Comparison to PriB, a C6 bacterial indole prenyltransferase, offers further insight regarding the structural basis of regioselective indole prenylation. Isothermal titration calorimetry measurements indicate a synergistic relationship between l-Trp and DMSPP binding. Finally, activity assays demonstrate the selectivity of CymD for l-Trp and indole as prenyl acceptors. Collectively, these data establish a foundation for understanding and engineering the regioselectivity of indole prenylation by members of the prenyltransferase protein family.

Reconstruction of tricarboxylic acid cycle in Corynebacterium glutamicum with a genome-scale metabolic network model for trans-4-hydroxyproline production.

trans-4-Hydroxy- l-proline (Hyp) is an abundant component of mammalian collagen and functions as a chiral synthon for the syntheses of anti-inflammatory drugs in the pharmaceutical industry. Proline 4-hydroxylase (P4H) can catalyze the conversion of l-proline to Hyp; however, it is still challenging for the fermentative production of Hyp from glucose using P4H due to the low yield and productivity. Here, we report the metabolic engineering of Corynebacterium glutamicum for the fermentative production of Hyp by reconstructing tricarboxylic acid (TCA) cycle together with heterologously expressing the p4h gene from Dactylosporangium sp. strain RH1. In silico model-based simulation showed that α-ketoglutarate was redirected from the TCA cycle toward Hyp synthetic pathway driven by P4H when the carbon flux from succinyl-CoA to succinate descended to zero. The interruption of the TCA cycle by the deletion of sucCD-encoding the succinyl-CoA synthetase (SUCOAS) led to a 60% increase in Hyp production and had no obvious impact on the growth rate. Fine-tuning of plasmid-borne ProB* and P4H abundances led to a significant increase in the yield of Hyp on glucose. The final engineered Hyp-7 strain produced up to 21.72 g/L Hyp with a yield of 0.27 mol/mol (Hyp/glucose) and a volumetric productivity of 0.36 g·L -1 ·hr -1 in the shake flask fermentation. To our knowledge, this is the highest yield and productivity achieved by microbial fermentation in a glucose-minimal medium for Hyp production. This strategy provides new insights into engineering C. glutamicum by flux coupling for the fermentative production of Hyp and related products.

Preparation, Assay, and Application of Chlorinase SalL for the Chemoenzymatic Synthesis of S-Adenosyl-l-Methionine and Analogs.

S-adenosyl-l-methionine (SAM) is universal in biology, serving as the second most common cofactor in a variety of enzymatic reactions. One of the main roles of SAM is the methylation of nucleic acids, proteins, and metabolites. Methylation often imparts regulatory control to DNA and proteins, and leads to an increase in the activity of specialized metabolites such as those developed as pharmaceuticals. There has been increased interest in using SAM analogs in methyltransferase-catalyzed modification of biomolecules. However, SAM and its analogs are expensive and unstable, degrading rapidly under physiological conditions. Thus, the availability of methods to prepare SAM in situ is desirable. In addition, synthetic methods to generate SAM analogs suffer from low yields and poor diastereoselectivity. The chlorinase SalL from the marine bacterium Salinispora tropica catalyzes the reversible, nucleophilic attack of chloride at the C5' ribosyl carbon of SAM leading to the formation of 5'-chloro-5'-deoxyadenosine (ClDA) with concomitant displacement of l-methionine. It has been demonstrated that the in vitro equilibrium of the SalL-catalyzed reaction favors the synthesis of SAM. In this chapter, we describe methods for the preparation of SalL, and the chemoenzymatic synthesis of SAM and SAM analogs from ClDA and l-methionine congeners using SalL. In addition, we describe procedures for the in situ chemoenzymatic synthesis of SAM coupled to DNA, peptide, and metabolite methylation, and to the incorporation of isotopes into alkylated products.

Construction of a mutant of Actinoplanes sp. N902-109 that produces a new rapamycin analog.

In the present study, we introduced point mutations into Ac_rapA which encodes a polyketide synthase responsible for rapamycin biosynthesis in Actinoplanes sp. N902-109, in order to construct a mutant with an inactivated enoylreductase (ER) domain, which was able to synthesize a new rapamycin analog. Based on the homologous recombination induced by double-strand breaks in chromosome mediated by endonuclease I-SceI, the site-directed mutation in the first ER domain of Ac_rapA was introduced using non-replicating plasmid pLYERIA combined with an I-SceI expression plasmid. Three amino acid residues of the active center, Ala-Gly-Gly, were converted to Ala-Ser-Pro. The broth of the mutant strain SIPI-027 was analyzed by HPLC and a new peak with the similar UV spectrum to that of rapamycin was found. The sample of the new peak was prepared by solvent extraction, column chromatography, and crystallization methods. The structure of new compound, named as SIPI-rapxin, was elucidated by determining and analyzing its MS and NMR spectra and its biological activity was assessed using mixed lymphocyte reaction (MLR). An ER domain-deficient mutant of Actinoplanes sp. N902-109, named as SIPI-027, was constructed, which produced a novel rapamycin analog SIPI-rapxin and its structure was elucidated to be 35, 36-didehydro-27-O-demethylrapamycin. The biological activity of SIPI-rapxin was better than that of rapamycin. In conclusion, inactivation of the first ER domain of rapA, one of the modular polyketide synthase responsible for macro-lactone synthesis of rapamycin, gave rise to a mutant capable of producing a novel rapamycin analog, 35, 36-didehydro-27-O-demethylrapamycin, demonstrating that the enoylreductase domain was responsible for the reduction of the double bond between C-35 and C-36 during rapamycin synthesis.

Deciphering Nature's Intricate Way of N,S-Dimethylating l-Cysteine: Sequential Action of Two Bifunctional Adenylation Domains.

Dimethylation of amino acids consists of an interesting and puzzling series of events that could be achieved, during nonribosomal peptide biosynthesis, either by a single adenylation (A) domain interrupted by a methyltransferase (M) domain or by the sequential action of two of such independent enzymes. Herein, to establish the method by which Nature N,S-dimethylates l-Cys, we studied its formation during thiochondrilline A biosynthesis by evaluating TioS(A3aM3SA3bT3) and TioN(AaMNAb). This study not only led to identification of the exact pathway followed in Nature by these two enzymes for N,S-dimethylation of l-Cys, but also revealed that a single interrupted A domain can N,N-dimethylate amino acids, a novel phenomenon in the nonribosomal peptide field. These findings offer important and useful insights for the development and engineering of novel interrupted A domain enzymes to serve, in the future, as tools for combinatorial biosynthesis.

Minimization of the Thiolactomycin Biosynthetic Pathway Reveals that the Cytochrome P450 Enzyme TlmF Is Required for Five-Membered Thiolactone Ring Formation.

Thiolactomycin (TLM) belongs to a class of rare and unique thiotetronate antibiotics that inhibit bacterial fatty acid synthesis. Although this group of natural product antibiotics was first discovered over 30 years ago, the study of TLM biosynthesis remains in its infancy. We recently discovered the biosynthetic gene cluster (BGC) for TLM from the marine bacterium Salinispora pacifica CNS-863. Here, we report the investigation of TLM biosynthetic logic through mutagenesis and comparative metabolic analyses. Our results revealed that only four genes (tlmF, tlmG, tlmH, and tlmI) are required for the construction of the characteristic γ-thiolactone skeleton of this class of antibiotics. We further showed that the cytochrome P450 TlmF does not directly participate in sulfur insertion and C-S bond formation chemistry but rather in the construction of the five-membered thiolactone ring as, upon its deletion, we observed the alternative production of the six-membered δ-thiolactomycin. Our findings pave the way for future biochemical investigation of the biosynthesis of this structurally unique group of thiotetronic acid natural products.

Structure of OxyA tei: completing our picture of the glycopeptide antibiotic producing Cytochrome P450 cascade.

Cyclization of glycopeptide antibiotic precursors occurs in either three or four steps catalyzed by Cytochrome P450 enzymes. Three of these enzymes have been structurally characterized to date with the second enzyme along the pathway, OxyA, escaping structural analysis. We are now able to present the structure of OxyAtei involved in teicoplanin biosynthesis - the same enzyme recently shown to be the first active OxyA homolog. In spite of the hydrophobic character of the teicoplanin precursor, the polar active site of OxyAtei and its affinity for certain azole inhibitors hint at its preference for substrates with polar decorations.

Discovery a novel organic solvent tolerant esterase from Salinispora arenicola CNP193 through genome mining.

An esterase gene, encoding a 325-amino-acid protein (SAestA), was mined form obligate marine actinomycete strain Salinispora arenicola CNP193 genome sequence. Phylogenetic analysis of the deduced amino acid sequence showed that the enzyme belonged to the family IV of lipolytic enzymes. The gene was cloned, expressed in Escherichia coli as a His-tagged protein, purified and characterized. The molecular weight of His-tagged SAestA is ∼38 kDa. SAestA-His6 was active in a temperature (5-40 °C) and pH range (7.0-11.0), and maximal activity was determined at pH 9.0 and 30 °C. The activity was severely inhibited by Hg(2+), Cu(2+), and Zn(2+). In particular, this enzyme showed remarkable stability in presence of organic solvents (25%, v/v) with log P>2.0 even after incubation for 7 days. All these characteristics suggested that SAestA may be a potential candidate for application in industrial processes in aqueous/organic media.

Substrate specificity and biochemical characterization of an adenylation domain from an obligate marine actinomycete.

Salinispora arenicola CNS-205 was a first-isolated obligate marine actinomycete. A gene (sare0357), annotated as ''amino acid adenylation domain'' located on the genome of Salinispora arenicola CNS-205, was cloned and characterized. The recombinant target protein Sare0357 was expressed in E. coli. Sare0357 specifically recognized and activated tryptophan (Trp) and phenylalanine (Phe). The basic kinetic parameters of Sare0357 for Trp were K m = 0.04 mM, V max = 2.1 µM/min, k cat = 14.2 min(-1), and for Phe were K m = 0.03 mM, V max = 1.6 µM/min, kcat = 10.4 min(-1). Our data elucidated Sare0357 biological role and biochemical properties as a Trp and Phe-activating adenylation domain.

Harnessing the synthetic capabilities of glycopeptide antibiotic tailoring enzymes: characterization of the UK-68,597 biosynthetic cluster.

In this study, a draft genome sequence of Actinoplanes sp. ATCC 53533 was assembled, and an 81-kb biosynthetic cluster for the unusual sulfated glycopeptide UK-68,597 was identified. Glycopeptide antibiotics are important in the treatment of infections caused by Gram-positive bacteria. Glycopeptides contain heptapeptide backbones that are modified by many tailoring enzymes, including glycosyltransferases, sulfotransferases, methyltransferases, and halogenases, generating extensive chemical and functional diversity. Several tailoring enzymes in the cluster were examined in vitro for their ability to modify glycopeptides, resulting in the synthesis of novel molecules. Tailoring enzymes were also expressed in the producer of the glycopeptide aglycone A47934, generating additional chemical diversity. This work characterizes the biosynthetic program of UK-68,597 and demonstrates the capacity to expand glycopeptide chemical diversity by harnessing the unique chemistry of tailoring enzymes.

Identification of fluorinases from Streptomyces sp MA37, Norcardia brasiliensis, and Actinoplanes sp N902-109 by genome mining.

The fluorinase is an enzyme that catalyses the combination of S-adenosyl-L-methionine (SAM) and a fluoride ion to generate 5'-fluorodeoxy adenosine (FDA) and L-methionine through a nucleophilic substitution reaction with a fluoride ion as the nucleophile. It is the only native fluorination enzyme that has been characterised. The fluorinase was isolated in 2002 from Streptomyces cattleya, and, to date, this has been the only source of the fluorinase enzyme. Herein, we report three new fluorinase isolates that have been identified by genome mining. The novel fluorinases from Streptomyces sp. MA37, Nocardia brasiliensis, and an Actinoplanes sp. have high homology (80-87 % identity) to the original S. cattleya enzyme. They all possess a characteristic 21-residue loop. The three newly identified genes were overexpressed in E. coli and shown to be fluorination enzymes. An X-ray crystallographic study of the Streptomyces sp. MA37 enzyme demonstrated that it is almost identical in structure to the original fluorinase. Culturing of the Streptomyces sp. MA37 strain demonstrated that it not only also elaborates the fluorometabolites, fluoroacetate and 4-fluorothreonine, similar to S. cattleya, but this strain also produces a range of unidentified fluorometabolites. These are the first new fluorinases to be reported since the first isolate, over a decade ago, and their identification extends the range of fluorination genes available for fluorination biotechnology.

Catalysis by desolvation: the catalytic prowess of SAM-dependent halide-alkylating enzymes.

In the biological fixation of halide ions, several enzymes have been found to catalyze alkyl transfer from S-adenosylmethionine to halide ions. It proves possible to measure the rates of reaction of the trimethylsulfonium ion with I(-), Br(-), Cl(-), F(-), HO(-), and H2O in water at elevated temperatures. Comparison of the resulting second-order rate constants, extrapolated to 25 °C, with the values of k(cat)/K(m) reported for fluorinase and chlorinase indicates that these enzymes enhance the rates of alkyl halide formation by factors of 2 × 10(15)- and 1 × 10(17)-fold, respectively. These rate enhancements, achieved without the assistance of cofactors, metal ions, or general acid-base catalysis, are the largest that have been reported for an enzyme that acts on two substrates.

Functional identification of the gene encoding the enzyme involved in mannosylation in ramoplanin biosynthesis in Actinoplanes sp.

Ramoplanin is a lipopeptide antibiotic active against multi-drug-resistant, Gram-positive pathogens. Structurally, it contains a di-mannose moiety attached to the peptide core at Hpg(11). The biosynthetic gene cluster of ramoplanin has already been reported and the assembly of the depsipeptide has been elucidated but the mechanism of transferring sugar moiety to the peptide core remains unclear. Sequence analysis of the biosynthetic gene cluster indicated ramo-orf29 was a mannosyltransferase candidate. To investigate the involvement of ramo-orf29 in ramoplanin biosynthesis, gene inactivation and complementation have been conducted in Actinoplanes sp. ATCC 33076 by homologous recombination. Metabolite analysis revealed that the ramo-orf29 inactivated mutant produced no ramoplanin but the ramoplanin aglycone. Thus, ramo-orf29 codes for the mannosyltransferase in the ramoplanin biosynthesis pathway. This lays the foundation for further exploitation of the ramoplanin mannosyltransferase and aglycone in combinatorial biosynthesis.

Production of ramoplanin analogues by genetic engineering of Actinoplanes sp.

Ramoplanins are lipopeptides effective against a wide range of Gram-positive pathogens. Ramoplanin A2 is in Phase III clinical trials. The structure-activity relationship of the unique 2Z,4E-fatty acid side-chain of ramoplanins indicates a significant contribution to the antimicrobial activities but ramoplanin derivatives with longer 2Z,4E-fatty acid side-chains are not easy to obtain by semi-synthetic approaches. To construct a strain that produces such analogues, an acyl-CoA ligase gene in a ramoplanin-producing Actinoplanes was inactivated and a heterologous gene from an enduracidin producer (Streptomyces fungicidicus) was introduced into the mutant. The resulting strain produced three ramoplanin analogues with longer alkyl chains, in which X1 was purified. The MIC value of X1 was ~0.12 µg/ml against Entrococcus sp. and was also active against vancomycin-resistant Staphylococcus aureus (MIC = 2 µg/ml).

Bioactivity-guided genome mining reveals the lomaiviticin biosynthetic gene cluster in Salinispora tropica.

The use of genome sequences has become routine in guiding the discovery and identification of microbial natural products and their biosynthetic pathways. In silico prediction of molecular features, such as metabolic building blocks, physico-chemical properties or biological functions, from orphan gene clusters has opened up the characterization of many new chemo- and genotypes in genome mining approaches. Here, we guided our genome mining of two predicted enediyne pathways in Salinispora tropica CNB-440 by a DNA interference bioassay to isolate DNA-targeting enediyne polyketides. An organic extract of S. tropica showed DNA-interference activity that surprisingly was not abolished in genetic mutants of the targeted enediyne pathways, ST_pks1 and spo. Instead we showed that the product of the orphan type II polyketide synthase pathway, ST_pks2, is solely responsible for the DNA-interfering activity of the parent strain. Subsequent comparative metabolic profiling revealed the lomaiviticins, glycosylated diazofluorene polyketides, as the ST_pks2 products. This study marks the first report of the 59 open reading frame lomaiviticin gene cluster (lom) and supports the biochemical logic of their dimeric construction through a pathway related to the kinamycin monomer.

A tandem chemoenzymatic methylation by S-adenosyl-L-methionine.

Keep 'em methylated: The in situ preparation of the cofactor AdoMet was achieved by allowing the biosynthetic enzyme SalL to operate in the reverse direction by presentation of 5'-chloro-5'-deoxyadenosine at low salt concentrations. This reaction was readily coupled with DNA and small molecule methyltransferases to afford a regioselective method for chemo-enzymatic methylation and isotope incorporation.

Versatility of enzymes catalyzing late steps in polyene 67-121C biosynthesis.

Actinoplanes caeruleus produces 67-121C, a heptaene macrolide modified with a D-mannosyl-D-mycosaminyl disaccharide. Draft genome sequencing revealed genes encoding mycosaminyltransferase, mycosamine synthase, a cytochrome P450 that modifies the macrolactone core, and the extending mannosyltransferase. Only the mycosamine synthase and P450 were active in the biosynthesis of amphotericins in Streptomyces nodosus, the amphotericin producer.

Characterization of sterol glucosyltransferase from Salinispora tropica CNB-440: potential enzyme for the biosynthesis of sitosteryl glucoside.

A sterol glucosyltransferase-encoded gene was isolated from Salinispora tropica CNB-440, a marine, sediment-dwelling, Gram positive bacterium that produces the potent anticancer compound, salinosporamide A. The full-length gene consists of 1284 nucleotides and encodes 427 amino acids with a calculated mass of 45.65kDa. The gene was then cloned and heterologously expressed in Escherichia coli BL21(DE3). The amino acid sequence shares 39% similarity with the glycosyltransferase from Withania somnifera, which belongs to glycosyltransferase family 1. Enzyme reactions were carried out with the various free sterols (acceptor) and NDP-sugars (donor). The purified protein only showed activity for glucosylation of ß-sitosterol with UDP-D-glucose and TDP-D-glucose donors, and optimal activity at pH 7.5 and 37°C. Among these two donors, UDP-D-glucose was preferred.