11 research outputs found

    Nature versus nurture in two highly enantioselective esterases from Bacillus cereus and Thermoanaerobacter tengcongensis

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    There is an increasing need for the use of biocatalysis to obtain enantiopure compounds as chiral building blocks for drug synthesis such as antibiotics. The principal findings of this study are: i) the complete sequenced genomes of Bacillus cereus ATCC 14579 and Thermoanaerobacter tengcongensis MB4 contain a hitherto undescribed enantioselective and alkaliphilic esterase (BcEST and TtEST, respectively) that is specific for the production of (R)-2-benzyloxy-propionic acid ethyl ester, a key intermediate in the synthesis of levofloxacin, a potent antibiotic; and, ii) directed evolution targeted for increased thermostability of BcEST produced two improved variants, but in either case the 3 \u2013 5\ub0C increase in the apparent melting temperature (Tm) of the mutants over the native BcEST that has a Tm of 50\ub0C was outperformed by TtEST, a naturally-occurring homolog with a Tm of 65\ub0C. Protein modeling of BcEST mapped the S148C and K272R mutations at protein surface and the I88T and Q110L mutations at more buried locations. This work expands the repertoire of characterized members of the \u3b1/\u3b2 fold-hydrolase superfamily. Further, it shows that genome mining is an economical option for new biocatalyst discovery and we provide a rare example of a naturally-occurring thermostable biocatalyst that outperforms experimentally-evolved homologs that carry out the same hydrolysis.Peer reviewed: YesNRC publication: Ye

    Thermostable feruloyl esterase for the bioproduction of ferulic acid from triticale bran

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    A putative \u3b1/\u3b2 hydrolase fold-encoding gene (locus tag TTE1809) from the genome of Thermoanaerobacter tengcongensis was cloned and expressed in Escherichia coli as a possible source of thermostable feruloyl esterase (FAE) for the production of antioxidant phenolic acids from biomass. Designated as TtFAE, the 33-kDa protein was purified to apparent homogeneity. The lipase-like sequence characteristics of TtFAE and its substrate specificity towards methyl ferulate, methyl sinapate, and methyl p-coumarate classify it as a new member of the type A FAEs. At 75\ub0C, the enzyme retained at least 95% of its original activity for over 80 min; at 80\ub0C, its half-life was found to be 50 min, rendering TtFAE a highly thermostable protein. Under different hydrolytic conditions, ferulic acid (FA) was shown to be released from feruloylated oligosaccharides prepared from triticale bran. An estimated recovery of 68 mg FA/100 g triticale bran was demonstrated by a 30% release of the total FA from triticale bran within a 5-h incubation period. Both the oxygen radical absorbing capacity values of the feruloylated oligosaccharides and free FA were also determined. Overall, this work introduces a new bacterial member to the growing family of plant cell wall degrading FAEs that at present is largely of fungal origin, and it benchmarks the bioproduction of FA from triticale bran.Peer reviewed: YesNRC publication: Ye

    Structural analysis of Bacillus pumilus phenolic acid decarboxylase, a lipocalin-fold enzyme

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    Acta Crystallographica Section F: Structural Biology and Crystallization Communicationsis a rapid all-electronic journal, which provides a home for short communications onthe crystallization and structure of biological macromolecules. It includes four categoriesof publication: protein structure communications; nucleic acid structure communications;structural genomics communications; and crystallization communications. Structuresdetermined through structural genomics initiatives or from iterative studies suchas those used in the pharmaceutical industry are particularly welcomed. Section F isessential for all those interested in structural biology including molecular biologists, biochemists,crystallization specialists, structural biologists, biophysicists, pharmacologistsand other life scientists.Acta Crystallographica Section F: Structural Biology and Crystallization Communications est une revue enti\ue8rement \ue9lectronique \ue0 parution rapide renfermant de courtes communications sur la cristallisation et la structure de macromol\ue9cules biologiques. Elle se divise en quatre cat\ue9gories de communications, soit la structure des prot\ue9ines, la structure des acides nucl\ue9iques, la g\ue9nomique structurale et la cristallisation. Les structures d\ue9termin\ue9es par des initiatives en g\ue9nomique structurale ou \ue0 partir d\u2019\ue9tudes it\ue9ratives, comme celles employ\ue9es dans l\u2019industrie pharmaceutique, sont particuli\ue8rement bien accueillies. Cette revue constitue un incontournable pour tous ceux et celles qui s\u2019int\ue9ressent \ue0 la biologie structurale, soit les biologistes mol\ue9culaires, les biochimistes, les sp\ue9cialistes de la cristallisation, les biologistes structuraux, les biophysiciens, les pharmacologues et autres chercheurs en sciences de la vie.Peer reviewed: YesNRC publication: Ye

    A flax-retting endopolygalacturonase-encoding gene from Rhizopus oryzae

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    A polygalacturonase from the filamentous fungus Rhizopus oryzae strain sb (NRRL 29086), previously shown to be effective in the retting of flax fibers, was shown by the analysis of its reaction products on polygalacturonic acid to be an endo-type. By zymogram analysis, the enzyme in the crude culture filtrate appeared as two active species of 37 and 40 kD. The endopolygalacturonase-encoding gene was cloned in Escherichia coli and its translated 383-amino acid sequence found to be identical to that of a presumed exopolygalacturonase found in R. oryzae strain YM9901 and 96% identical to a hypothetical protein (RO3G_04731.1) in the sequenced genome of R. oryzae strain 99-880. Phylogenetic analysis revealed the presence of an unique cluster of Rhizopus polygalacturonase sequences that are separate from other fungal polygalacturonases. Conservation of 12 cysteines appears to be a special feature of this family of Rhizopus polygalacturonase sequences.NRC publication: Ye

    Camphor pathway redux: functional recombinant expression of 2,5-and 3,6-diketocamphane monooxygenases of Pseudomonas putida ATCC 17453 with their cognate flavin reductase catalyzing Baeyer-Villiger reactions

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    Whereas the biochemical properties of the monooxygenase components that catalyze the oxidation of 2,5-diketocamphane and 3,6-diketocamphane (2,5-DKCMO and 3,6-DKCMO, respectively) in the initial catabolic steps of (+) and (-) isomeric forms of camphor (CAM) metabolism in Pseudomonas putida ATCC 17453 are relatively well characterized, the actual identity of the flavin reductase (Fred) component that provides the reduced flavin to the oxygenases has hitherto been ill defined. In this study, a 37-kDa Fred was purified from a camphor-induced culture of P. putida ATCC 17453 and this facilitated cloning and characterization of the requisite protein. The active Fred is a homodimer with a subunit molecular weight of 18,000 that uses NADH as an electron donor (Km(32 \u3bcM), and it catalyzes the reduction of flavin mononucleotide (FMN) (Km=3.6 \u3bcM; kcat=283 s-\ub9) in preference to flavin adenine dinucleotide (FAD) (Km(19 \u3bcM; kcat(128 s-\ub9). Sequence determination of~40 kb of the CAM degradation plasmid revealed the locations of two isofunctional 2,5-DKCMO genes (camE25-1 for 2,5-DKCMO-1 and camE25-2 for 2,5-DKCMO-2) as well as that of a 3,6-DKCMO-encoding gene (camE36). In addition, by pulsed-field gel electrophoresis, the CAM plasmid was established to be linear and~533 kb in length. To enable functional assessment of the two-component monooxygenase system in Baeyer-Villiger oxidations, recombinant plasmids expressing Fred in tandem with the respective 2,5-DKCMO- and 3,6-DKCMO-encoding genes in Escherichia coli were constructed. Comparative substrate profiling of the isofunctional 2,5-DCKMOs did not yield obvious differences in Baeyer-Villiger biooxidations, but they are distinct from 3,6-DKCMO in the stereoselective oxygenations with various mono- and bicyclic ketone substrates.Peer reviewed: YesNRC publication: Ye

    Physiological, biochemical, and genetic characterization of an alicyclic amine-degrading Mycobacterium sp. strain THO100 isolated from a morpholine-containing culture of activated sewage sludge

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    Mycobacterium sp. strain THO100 was isolated from a morpholine-containing culture of activated sewage sludge. This strain was able to utilize pyrrolidine, morpholine, piperidine, piperazine, and 1,2,3,6-tetrahydropyridine as the sole sources of carbon, nitrogen, and energy. The degradation pathway of pyrrolidine as the best substrate for cellular growth was proposed based on the assays of substrate-induced cytochrome P450 and constitutive enzyme activities toward 4-aminobutyric acid (GABA) and succinic semialdehyde (SSA). Its 16S ribosomal RNA gene sequence (16S rDNA) was identical to that of Mycobacterium tokaiense ATCC 27282(T). The morABC genes responsible for alicyclic amine degradation were nearly identical among different species of Mycobacteria. Remarkably, repetitive sequences at the intergenic spacer (IGS) region between morC and orf1' were detected by comparison of the nearly identical mor gene cluster regions. Considering the strain activity for alicyclic amine degradation, the deleted 65-bp DNA segment did not significantly alter the open reading frames, and the expression and functions of the P450(mor) system remained unaltered. In addition, we found a spontaneous deletion of P450(mor) from another strain HE5 containing the archetypal mor gene cluster, which indicated a possible occurrence of DNA recombination to rearrange the DNA.NRC publication: Ye

    Characterization of the basic replicon of Rhodococcus plasmid pSOX and development of a Rhodococcus-Escherichia coli shuttle vector

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    The replication region of a 100-kb desulfurization plasmid (pSOX) from Rhodococcus sp. strain X309 was localized to a 4-kbKpnI fragment, and its sequence was determined. The amino acid sequence of one of the predicted open reading frames (ORFs) was related to the putative replication (Rep) protein sequences of the mycobacterial pLR7 family of plasmids. Three of the five predicted ORF products were identified by radiolabelling with the Escherichia coli T7 polymerase/promoter system. In E. coli, the Rep protein of pSOX was apparently synthesized in a shortened form, 21.3 kDa instead of the predicted 41.3 kDa, as a result of an internal initiation. This situation is reminescent of that for some bacterial Rep proteins. A shuttle plasmid was constructed with the pSOX origin, pBluescript II KS 12, and the chloramphenicol resistance (Cmr) gene from pRF29. This new shuttle plasmid was used to demonstrate expression of the Bacillus subtilis sacB gene in a strain of Rhodococcus, rendering it sensitive to the presence of sucrose.NRC publication: Ye

    Pseudomonad cyclopentadecanone monooxygenase displaying an uncommon spectrum of Baeyer-Villiger oxidations of cyclic ketones

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    Baeyer-Villiger monooxygenases (BVMOs) are biocatalysts that offer the prospect of high chemo-, regio-, and enantioselectivity in the organic synthesis of lactones or esters from a variety of ketones. In this study, we have cloned, sequenced, and overexpressed in Escherichia coli a new BVMO, cyclopentadecanone monooxygenase (CpdB or CPDMO), originally derived from Pseudomonas sp. strain HI-70. The 601-residue primary structure of CpdB revealed only 29% to 50% sequence identity to those of known BVMOs. A new sequence motif, characterized by a cluster of charged residues, was identified in a subset of BVMO sequences that contain an N-terminal extension of approximately 60 to 147 amino acids. The 64-kDa CPDMO enzyme was purified to apparent homogeneity, providing a specific activity of 3.94 micromol/min/mg protein and a 20% yield. CPDMO is monomeric and NADPH dependent and contains approximately 1 mol flavin adenine dinucleotide per mole of protein. A deletion mutant suggested the importance of the N-terminal 54 amino acids to CPDMO activity. In addition, a Ser261Ala substitution in a Rossmann fold motif resulted in an improved stability and increased affinity of the enzyme towards NADPH compared to the wild-type enzyme (K(m) = 8 microM versus K(m) = 24 microM). Substrate profiling indicated that CPDMO is unusual among known BVMOs in being able to accommodate and oxidize both large and small ring substrates that include C(11) to C(15) ketones, methyl-substituted C(5) and C(6) ketones, and bicyclic ketones, such as decalone and beta-tetralone. CPDMO has the highest affinity (K(m) = 5.8 microM) and the highest catalytic efficiency (k(cat)/K(m) ratio of 7.2 x 10(5) M(-1) s(-1)) toward cyclopentadecanone, hence the Cpd designation. A number of whole-cell biotransformations were carried out, and as a result, CPDMO was found to have an excellent enantioselectivity (E > 200) as well as 99% S-selectivity toward 2-methylcyclohexanone for the production of 7-methyl-2-oxepanone, a potentially valuable chiral building block. Although showing a modest selectivity (E = 5.8), macrolactone formation of 15-hexadecanolide from the kinetic resolution of 2-methylcyclopentadecanone using CPDMO was also demonstrated.NRC publication: Ye

    Crystal structures of cyclohexanone monooxygenase reveal complex domain movements and a sliding cofactor

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    Cyclohexanone monooxygenase (CHMO) is a flavoprotein that carries out the archetypical Baeyer 12Villiger oxidation of a variety of cyclic ketones into lactones. Using NADPH and O2 as cosubstrates, the enzyme inserts one atom of oxygen into the substrate in a complex catalytic mechanism that involves the formation of a flavin-peroxide and Criegee intermediate. We present here the atomic structures of CHMO from an environmental Rhodococcus strain bound with FAD and NADP+ in two distinct states, to resolutions of 2.3 and 2.2 \uc5. The two conformations reveal domain shifts around multiple linkers and loop movements, involving conserved arginine 329 and tryptophan 492, which effect a translation of the nicotinamide resulting in a sliding cofactor. Consequently, the cofactor is ideally situated and subsequently repositioned during the catalytic cycle to first reduce the flavin and later stabilize formation of the Criegee intermediate. Concurrent movements of a loop adjacent to the active site demonstrate how this protein can effect large changes in the size and shape of the substrate binding pocket to accommodate a diverse range of substrates. Finally, the previously identified BVMO signature sequence is highlighted for its role in coordinating domain movements. Taken together, these structures provide mechanistic insights into CHMO-catalyzed Baeyer 12Villiger oxidation.NRC publication: Ye
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