Myosin-cross-reactive antigen (MCRA) protein from <Emphasis Type="Italic">Bifidobacterium breve</Emphasis> is a FAD-dependent fatty acid hydratase which has a function in stress protection

Background  

The aim of this study was to determine the catalytic activity and physiological role of myosin-cross-reactive antigen (MCRA) from Bifidobacterium breve NCIMB 702258. MCRA from B. breve NCIMB 702258 was cloned, sequenced and expressed in heterologous hosts (Lactococcus and Corynebacterium) and the recombinant proteins assessed for enzymatic activity against fatty acid substrates.     

Knowledge-based design of bimodular and trimodular polyketide synthases based on domain and module swaps: a route to simple statin analogues.

BACKGROUND: Polyketides are structurally diverse natural products that have a range of medically useful activities. Nonaromatic bacterial polyketides are synthesised on modular polyketide synthase (PKS) multienzymes, in which each cycle of chain extension requires a different 'module' of enzymatic activities. Attempts to design and construct modular PKSs that synthesise specified novel polyketides provide a particularly stringent test of our understanding of PKS structure and function. RESULTS: We have constructed bimodular and trimodular PKSs based on DEBS1-TE, a derivative of the erythromycin PKS that contains only modules 1 and 2 and a thioesterase (TE), by substituting multiple domains with appropriate counterparts derived from the rapamycin PKS. Hybrid PKSs were obtained that synthesised the predicted target triketide lactones, which are simple analogues of cholesterol-lowering statins. In constructing intermodular fusions, whether between modules in the same or in different proteins, it was found advantageous to preserve intact the acyl carrier protein-ketosynthase (ACP-KS) didomain that spans the junction between successive modules. CONCLUSIONS: Relatively simple considerations govern the construction of functional hybrid PKSs. Fusion sites should be chosen either in the surface-accessible linker regions between enzymatic domains, as previously revealed, or just inside the conserved margins of domains. The interaction of an ACP domain with the adjacent KS domain, whether on the same polyketide or not, is of particular importance, both through conservation of appropriate protein-protein interactions, and through optimising molecular recognition of the altered polyketide chain in the key transfer of the acyl chain from the ACP of one module to the KS of the downstream module.     

Acyl‐Chain Elongation Drives Ketosynthase Substrate Selectivity in trans‐Acyltransferase Polyketide Synthases

Type I modular polyketide synthases (PKSs), which are responsible for the biosynthesis of many biologically active agents, possess a ketosynthase (KS) domain within each module to catalyze chain elongation. Acylation of the KS active site Cys residue is followed by transfer to malonyl‐ACP to yield an extended β‐ketoacyl chain (ACP=acyl carrier protein). To date, the precise contribution of KS selectivity in controlling product fidelity has been unclear. Six KS domains from trans‐acyltransferase (trans‐AT) PKSs were subjected to a mass spectrometry based elongation assay, and higher substrate selectivity was identified for the elongating step than in preceding acylation. A close correspondence between the observed KS selectivity and that predicted by phylogenetic analysis was seen. These findings provide insights into the mechanism of KS selectivity in this important group of PKSs, can serve as guidance for engineering, and show that targeted mutagenesis can be used to expand the repertoire of acceptable substrates.     

Evidence for a protein-protein interaction motif on an acyl carrier protein domain from a modular polyketide synthase

During biosynthesis on modular polyketide synthases (PKSs), chain extension intermediates are tethered to acyl carrier protein (ACP) domains through phosphopantetheinyl prosthetic groups. Each ACP must therefore interact with every other domain within the module, and also with a downstream acceptor domain. The nature of these interactions is key to our understanding of the topology and operation of these multienzymes. Sequence analysis and homology modeling implicates a potential helical region (helix II) on the ACPs as a protein-protein interaction motif. Using site-directed mutagenesis, we show that residues along this putative helix lie at the interface between the ACP and the phosphopantetheinyl transferase that catalyzes its activation. Our results accord with previous studies of discrete ACP proteins from fatty acid and aromatic polyketide biosynthesis, suggesting that helix II may also serve as a universal interaction motif in modular PKSs.     

Control Mechanism for cis Double‐Bond Formation by Polyunsaturated Fatty‐Acid Synthases

Polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and arachidonic acid (ARA) are essential fatty acids for humans. Some microorganisms biosynthesize these PUFAs through PUFA synthases composed of four subunits with multiple catalytic domains. These PUFA synthases each create a specific PUFA without undesirable byproducts, even though the multiple catalytic domains in each large subunit are very similar. However, the detailed biosynthetic pathways and mechanisms for controlling final‐product profiles are still obscure. In this study, the FabA‐type dehydratase domain (DH_FabA) in the C‐subunit and the polyketide synthase‐type dehydratase domain (DH_PKS) in the B‐subunit of ARA synthase were revealed to be essential for ARA biosynthesis by in vivo gene exchange assays. Furthermore, in vitro analysis with truncated recombinant enzymes and C₄‐ to C₈‐acyl ACP substrates showed that ARA and EPA synthases utilized two types of DH domains, DH_PKS and DH_FabA, depending on the carbon‐chain length, to introduce either saturation or cis double bonds to growing acyl chains.     

Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the <Emphasis Type="Italic">Aedes aegypti</Emphasis> Mosquito Utilizing <Emphasis Type="Italic">E. coli</Emphasis> as a host

Background

Studying proteins and enzymes involved in important biological processes in the Aedes aegypti mosquito is limited by the quantity that can be directly isolated from the mosquito. Adding to this difficulty, digestive enzymes (midgut proteases) involved in metabolizing blood meal proteins require a more oxidizing environment to allow proper folding of disulfide bonds. Therefore, recombinant techniques to express foreign proteins in Escherichia coli prove to be effective in producing milligram quantities of the expressed product. However, with the most commonly used strains having a reducing cytoplasm, soluble expression of recombinant proteases is hampered. Fortunately, new E. coli strains with a more oxidizing cytoplasm are now available to ensure proper folding of disulfide bonds.

Results

Utilizing an E. coli strain with a more oxidizing cytoplasm (SHuffle® T7, New England Biolabs) and changes in bacterial growth temperature has resulted in the soluble expression of the four most abundantly expressed Ae. aegypti midgut proteases (AaET, AaSPVI, AaSPVII, and AaLT). A previous attempt of solubly expressing the full-length zymogen forms of these proteases with the leader (signal) sequence and a modified pseudo propeptide with a heterologous enterokinase cleavage site led to insoluble recombinant protein expression. In combination with the more oxidizing cytoplasm, and changes in growth temperature, helped improve the solubility of the zymogen (no leader) native propeptide proteases in E. coli. Furthermore, the approach led to autocatalytic activation of the proteases during bacterial expression and observable BApNA activity. Different time-points after bacterial growth induction were tested to determine the time at which the inactive (zymogen) species is observed to transition to the active form. This helped with the purification and isolation of only the inactive zymogen forms using Nickel affinity.

Conclusions

The difficulty in solubly expressing recombinant proteases in E. coli is caused by the native reducing cytoplasm. However, with bacterial strains with a more oxidizing cytoplasm, recombinant soluble expression can be achieved, but only in concert with changes in bacterial growth temperature. The method described herein should provide a facile starting point to recombinantly expressing Ae. aegypti mosquito proteases or proteins dependent on disulfide bonds utilizing E. coli as a host.

     

Comparison of sterols and fatty acids in two species of <Emphasis Type="Italic">Ganoderma</Emphasis>

Background  

Two species of Ganoderma, G. sinense and G. lucidum, are used as Lingzhi in China. Howerver, the content of triterpenoids and polysaccharides, main actives compounds, are significant different, though the extracts of both G. lucidum and G. sinense have antitumoral proliferation effect. It is suspected that other compounds contribute to their antitumoral activity. Sterols and fatty acids have obvious bioactivity. Therefore, determination and comparison of sterols and fatty acids is helpful to elucidate the active components of Lingzhi.     

Thermophile-specific proteins: the gene product of <Emphasis Type="Italic">aq_1292</Emphasis> from <Emphasis Type="Italic">Aquifex aeolicus</Emphasis> is an NTPase

Background  

To identify thermophile-specific proteins, we performed phylogenetic patterns searches of 66 completely sequenced microbial genomes. This analysis revealed a cluster of orthologous groups (COG1618) which contains a protein from every thermophile and no sequence from 52 out of 53 mesophilic genomes. Thus, COG1618 proteins belong to the group of thermophile-specific proteins (THEPs) and therefore we here designate COG1618 proteins as THEP1s. Since no THEP1 had been analyzed biochemically thus far, we characterized the gene product of aq_1292 which is THEP1 from the hyperthermophilic bacterium Aquifex aeolicus (aaTHEP1).     

Role of the linker region in the expression of <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> glucoamylase

Background  

Rhizopus oryzaeglucoamylase (RoGA) consists of three domains: an amino (N)-terminal raw starch-binding domain (SBD), a glycosylated linker domain, and a carboxy (C)-terminal catalytic domain. The 36-amino-acid linker region (residues 132–167) connects the two functional domains, but its structural and functional roles are unclear.     

Identification of Claisen cyclase domain in fungal polyketide synthase WA, a naphthopyrone synthase of Aspergillus nidulans

BACKGROUND: Based on the homology with fatty acid synthases and bacterial polyketide synthases (PKSs), thioesterase domains have been assigned at the C-terminus regions of fungal iterative type I PKSs. We previously overexpressed Aspergillus nidulans wA PKS gene in a heterologous fungal host and identified it to encode a heptaketide naphthopyrone synthase. In addition, expression of C-terminus-modified WA PKS gave heptaketide isocoumarins suggesting that the C-terminus region of WA PKS is involved in the cyclization of the second aromatic ring of naphthopyrone. To unravel the actual function of the C-terminus region, we carried out functional analysis of WA PKS mutants by C-terminus deletion and site-directed mutagenesis. RESULTS: Only the 32 amino acid deletion from the C-terminus of WA PKS caused product change to heptaketide isocoumarins from heptaketide naphthopyrone, YWA1 1, a product of intact WA PKS. Further C-terminus deletion mutant of WA PKS up to Ser(1967), an active site residue of so far called thioesterase, still produced isocoumarins. Site-directed mutagenesis of amino acid residues in this C-terminus region showed that even a single mutation of S1967A or H2129Q caused production of isocoumarin instead of naphthopyrone. Furthermore, the role of tandem acyl carrier proteins (ACPs), a typical feature of fungal aromatic PKSs, was examined by site-directed mutagenesis and the results indicated that both ACPs can function as ACP independently. CONCLUSIONS: Claisen-type cyclization is assumed to be involved in formation of aromatic compounds by some fungal type I PKSs. These PKSs have a quite identical architecture of active site domain organization, beta-ketoacyl synthase, acyltransferase, tandem ACPs and thioesterase (TE) domains. Since the C-terminus region of WA PKS of this type was determined to be involved in Claisen-type cyclization of the second ring of naphthopyrone, we propose that the so far called TE of these PKSs work not just as TE but as Claisen cyclase.     

Control Mechanism for Carbon‐Chain Length in Polyunsaturated Fatty‐Acid Synthases

Polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are essential fatty acids. PUFA synthases are composed of three to four subunits and each create a specific PUFA without undesirable byproducts. However, detailed biosynthetic mechanisms for controlling final product profiles have been obscure. Here, the bacterial DHA and EPA synthases were carefully dissected by in vivo and in vitro experiments. In vitro analysis with two KS domains (KS_A and KS_C) and acyl‐acyl carrier protein (ACP) substrates showed that KS_A accepted short‐ to medium‐chain substrates while KS_C accepted medium‐ to long‐chain substrates. Unexpectedly, condensation from C₁₈ to C₂₀, the last elongation step in EPA biosynthesis, was catalyzed by KS_A domains in both EPA and DHA synthases. Conversely, condensation from C₂₀ to C₂₂, the last elongation step for DHA biosynthesis, was catalyzed by the KS_C domain in DHA synthase. KS_C domains therefore determine the chain lengths.     

Reconstitution of a Type II Polyketide Synthase that Catalyzes Polyene Formation

While type II polyketide synthases (PKSs) are known for producing aromatic compounds, a phylogenetically new subfamily of type II PKSs have been recently proposed to synthesize polyene structures. Here we report in vitro analysis of such a type II PKS, IgaPKS for ishigamide biosynthesis. The ketoreductase (Iga13) and dehydratase (Iga16) were shown to catalyze the reduction of a β‐keto group and dehydration of a β‐hydroxy group, respectively, to form a trans double bond. Incubation of the acyl carrier protein (Iga10), the ketosynthase/chain length factor complex (Iga11–Iga12), Iga13 and Iga16 with malonyl and hexanoyl‐CoAs and NADPH followed by KOH hydrolysis resulted in the formation of four unsaturated carboxylic acids (C₈, C₁₀, C₁₂, and C₁₄), indicating that IgaPKS catalyzes tetraene formation by repeating the cycle of condensation, keto‐reduction and dehydration with strict stereo‐specificity. We propose “highly reducing type II PKS subfamily” for the polyene‐producing type II PKSs.     

Profiling of ornithine lipids in bacterial extracts of Rhodobacter sphaeroides by reversed-phase liquid chromatography with electrospray ionization and multistage mass spectrometry (RPLC-ESI-MS)

Ornithine lipids (OLs), a sub-group of the large (and of emerging interest) family of lipoamino acids of bacterial origin, contain a 3-hydroxy fatty acyl chain linked via an amide bond to the α-amino group of ornithine and via an ester bond to a second fatty acyl chain. OLs in extracts of Rhodobacter sphaeroides (R. sphaeroides) were investigated by high-performance reversed phase liquid chromatography (RPLC) with electrospray ionization mass spectrometry (ESI-MS) in negative ion mode using a linear ion trap (LIT). The presence of OLs bearing both saturated (i.e, 16:0, 17:0, 18:0, 19:0 and 20:0) and unsaturated chains (i.e., 18:1, 19:1, 19:2 and 20:1) was ascertained and their identification, even for isomeric, low abundance and partially co-eluting species, was achieved by low-energy collision induced dissociation (CID) multistage mass spectrometry (MSⁿ, n = 2–4). OLs signatures found in two R. sphaeroides strains, i.e., wild type 2.4.1 and mutant R26, were examined and up to 16 and 17 different OL species were successfully identified, respectively. OLs in both bacterial strains were characterized by several combinations of fatty chains on ester-linked and amide-linked 3-OH fatty acids. Multistage MS spectra of monoenoic amide-linked 3-OH acyl chains, allowed the identification of positional isomer of OL containing 18:1 (i.e. 9-octadecenoic) and 20:1 (i.e. 11-eicosenoic) fatty acids. The most abundant OL ([M−H]⁻ at m/z 717.5) in R. sphaeroides R26 was identified as OL 3-OH 20:1/19:1 (i.e., 3-OH-eicosenoic acid amide-linked to ornithine and esterified to a nonadecenoic chain containing a cyclopropane ring). An unusual OL (m/z 689.5 for the [M−H]⁻ ion), most likely containing a cyclopropene ester-linked acyl chain (i.e., OL 3-OH 18:0/19:2), was retrieved only in the carotenoidless mutant strain R26. Based on the biosynthetic pathways already known for cyclopropa(e)ne ring-including acyl chains, a plausible explanation was invoked for the enzymatic generation of this ester-linked chain in R. sphaeroides.     

Kinetic characterisation of arylamine <Emphasis Type="Italic">N</Emphasis>-acetyltransferase from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Background  

Arylamine N-acetyltransferases (NATs) are important drug- and carcinogen-metabolising enzymes that catalyse the transfer of an acetyl group from a donor, such as acetyl coenzyme A, to an aromatic or heterocyclic amine, hydrazine, hydrazide or N-hydroxylamine acceptor substrate. NATs are found in eukaryotes and prokaryotes, and they may also have an endogenous function in addition to drug metabolism. For example, NAT from Mycobacterium tuberculosis has been proposed to have a role in cell wall lipid biosynthesis, and is therefore of interest as a potential drug target. To date there have been no studies investigating the kinetic mechanism of a bacterial NAT enzyme.     

Pentavalent Arsenate Reductase Activity in Cytosolic Fractions of <Emphasis Type="Italic">Pseudomonas</Emphasis> sp., Isolated from Arsenic-Contaminated Sites of Tezpur,Assam

Pentavalent arsenate reductase activity was localized and characterized in vitro in the cytosolic fraction of a newly isolated bacterial strain from arsenic-contaminated sites. The bacterium was gram negative, rod-shaped, nonmotile, non-spore-forming, and noncapsulated, and the strain was identified as Pseudomonas sp. DRBS1 following biochemical and molecular approaches. The strain Pseudomonas sp. DRBS1 exhibited enzymatic machinery for reduction of arsenate(V) to arsenite(III). The suspended culture of the bacterium reduced more than 97% of As(V) (40–100 mM) to As(III) in 48 h. The growth rate and total cellular yield decreased in the presence of higher concentration of arsenate. The suspended culture repeatedly reduced 10 mM As(V) within 5 h up to five consecutive inputs. The cell-free extracts reduced 86% of 100 μM As(V) in 40 min. The specific activity of arsenate reductase enzyme in the presence of 100 μM arsenate is 6.68 μmol/min per milligram protein. The arsenate reductase activity is maximum at 30 °C and at pH 5.2. The arsenate reductase activity increased in the presence of electron donors like citrate, glucose, and galactose and metal ions like Cd⁺², Cu⁺², Ca⁺², and Fe⁺². Selenate as an electron donor also supports the growth of strain DRBS1 and significantly increased the arsenate reduction.     

Functional role of the additional domains in inulosucrase (IslA) from <Emphasis Type="Italic">Leuconostoc citreum</Emphasis> CW28

Background  

Inulosucrase (IslA) from Leuconostoc citreum CW28 belongs to a new subfamily of multidomain fructosyltransferases (FTFs), containing additional domains from glucosyltransferases. It is not known what the function of the additional domains in this subfamily is.     

Heterologous expression, purification, reconstitution and kinetic analysis of an extended type II polyketide synthase.

BACKGROUND: Polyketide synthases (PKSs) are bacterial multienzyme systems that synthesize a broad range of natural products. The 'minimal' PKS consists of a ketosynthase, a chain length factor, an acyl carrier protein and a malonyl transferase. Auxiliary components (ketoreductases, aromatases and cyclases are involved in controlling the oxidation level and cyclization of the nascent polyketide chain. We describe the heterologous expression and reconstitution of several auxiliary PKS components including the actinorhodin ketoreductase (act KR), the griseusin aromatase/cyclase (gris ARO/CYC), and the tetracenomycin aromatase/cyclase (tcm ARO/CYC). RESULTS: The polyketide products of reconstituted act and tcm PKSs were identical to those identified in previous in vivo studies. Although stable protein-protein interactions were not detected between minimal and auxiliary PKS components, kinetic analysis revealed that the extended PKS comprised of the act minimal PKS, the act KR and the gris ARO/CYC had a higher turnover number than the act minimal PKS plus the act KR or the act minimal PKS alone. Adding the tcm ARO/CYC to the tcm minimal PKS also increased the overall rate. CONCLUSIONS: Until recently the principal strategy for functional analysis of PKS subunits was through heterologous expression of recombinant PKSs in Streptomyces. Our results corroborate the implicit assumption that the product isolated from whole-cell systems is the dominant product of the PKS. They also suggest that an intermediate is channeled between the various subunits, and pave the way for more detailed structural and mechanistic analysis of these multienzyme systems.     

Purification and Kinetic Properties of Human Recombinant Dihydrofolate Reductase Produced in <Emphasis Type="Italic">Bombyx mori</Emphasis> Chrysalides

Recent reports describe the inhibition of human dihydrofolate reductase (hDHFR) by natural tea polyphenols. This finding could explain the epidemiologic data on their prophylactic effects for certain forms of cancer, and it raises the possibility that natural and synthetic polyphenols could be used in cancer chemotherapy. In order to obtain larger quantities of hDHFR to support structural studies, we established and validated a baculovirus system for the expression of this protein in Bombyx mori chrysalides (pupae of the silkworm enclosed in a cocoon). To isolate the expressed protein, whole infected pupae were homogenized, and the expressed protein was purified by affinity chromatography. Here, we demonstrate the efficient expression of recombinant hDHFR in this model and report that this newly expressed protein has high enzymatic activity and kinetic properties similar to those previously reported for recombinant hDHFR expressed in Escherichia coli. The purified protein showed dissociation constants for the binding of natural polyphenols similar to that expressed in E. coli, which ensures its usage as a new tool for further structural studies. Although the hDHFR yield per individual was found to be lower in the chrysalides than in the larvae of B. mori, the former system was optimized as a model for the scaled-up production of recombinant proteins. Expression of proteins in chrysalides (instead of larvae) could offer important advantages from both economic and biosecurity aspects.     

Polyketide proofreading by an acyltransferase-like enzyme

Trans-acyltransferase polyketide synthases (trans-AT PKSs) are an important group of bacterial enzymes producing bioactive polyketides. One difference from textbook PKSs is the presence of one or more free-standing AT-like enzymes. While one homolog loads the PKS with malonyl units, the function of the second copy (AT2) was unknown. We studied the two ATs PedC and PedD involved in pederin biosynthesis in an uncultivated symbiont. PedD displayed malonyl- but not acetyltransferase activity toward various acyl carrier proteins (ACPs). In contrast, the AT2 PedC efficiently hydrolyzed acyl units bound to N-acetylcysteamine or ACP. It accepted substrates with various chain lengths and functionalizations but did not cleave malonyl-ACP. These data are consistent with the role of PedC in?PKS proofreading, suggesting a similar function for other AT2 homologs and providing strategies for?polyketide titer improvement and biosynthetic investigations.     

Unprecedented mechanism of chain length determination in fungal aromatic polyketide synthases

Fungal aromatic polyketides show remarkable structural diversity fundamentally derived from variations in chain length and cyclization pattern. Their basic skeletons are synthesized by multifunctional iterative type I polyketide synthases (PKSs). Recently, we have found that the C-terminal thioesterase (TE)-like domain of Aspergillus nidulans WA catalyzes Claisen-type cyclization to form the B-ring of naphthopyrone YWA1. Here we report the unprecedented mechanism of chain length determination by the C-terminal TE-like domain of Colletotrichum lagenarium PKS1, which, in addition to catalyzing Claisen-type cyclization, intercepts the polyketomethylene intermediate from the acyl carrier protein domain during the condensation reaction to produce shorter chain length products. This chain length determination system is novel among PKSs, including bacterial and plant PKSs. The functional diversity of the TE-like domain directly influences the structural diversity of aromatic polyketides in C. lagenarium PKS1.