Biotransformation of L-lysine to L-pipecolic acid catalyzed by L-lysine 6-aminotransferase and pyrroline-5-carboxylate reductase

The enzyme involved in the reduction of delta1-piperideine-6-carboxylate (P6C) to L-pipecolic acid (L-PA) has never been identified. We found that Escherichia coli JM109 transformed with the lat gene encoding L-lysine 6-aminotransferase (LAT) converted L-lysine (L-Lys) to L-PA. This suggested that there is a gene encoding "P6C reductase" that catalyzes the reduction of P6C to L-PA in the genome of E. coli. The complementation experiment of proC32 in E. coli RK4904 for L-PA production clearly shows that the expression of both lat and proC is essential for the biotransformation of L-Lys to L-PA. Further, We showed that both LAT and pyrroline-5-carboxylate (P5C) reductase, the product of proC, were needed to convert L-Lys to L-PA in vitro. These results demonstrate that P5C reductase catalyzes the reduction of P6C to L-PA. Biotransformation of L-Lys to L-PA using lat-expressing E. coli BL21 was done and L-PA was accumulated in the medium to reach at an amount of 3.9 g/l after 159 h of cultivation. It is noteworthy that the ee-value of the produced pipecolic acid was 100%.     

Multicopy proC in Streptomyces coelicolor A3(2) elicits a transient production of prodiginines, while proC deletion does not yield a proline auxotroph

The last step of proline biosynthesis is typically catalysed by the enzyme Δ(1)-pyrroline-5-carboxylate reductase, encoded by the proC gene. Complete genome sequencing of Streptomyces coelicolor, a soil-dwelling Gram-positive bacterium that uses proline as a precursor for synthesis of prodiginine, revealed a single copy of this gene. Unexpectedly, disruption of this proC homologue (Sco3337) in S. coelicolor M145 yielded a prototrophic strain, yet the reductase activity of Sco3337 was confirmed by complementation of an Escherichia coli proC mutant. Multicopy proC within different genetic contexts elicited a transient production of prodiginines, which showed differential production kinetics of the two most common forms of this natural product produced by S. coelicolor, i.e. streptorubin B (cyclic) and undecylprodigiosin (linear). The metabolic and evolutionary implications of these observations are discussed.     

Escherichia coli delta 1-pyrroline-5-carboxylate reductase: gene sequence, protein overproduction and purification.

The sequence of the Escherichia coli proC gene which encodes for delta 1-pyrroline-5-carboxylate (PCA) reductase was determined. Overproduction of the proC gene product via an expression plasmid carrying the bacteriophage lambda PL promoter allowed the purification to homogeneity of PCA reductase by affinity adsorption chromatography. NH2 and COOH-terminal analysis and amino acid composition of the purified proC protein is consistent with the gene sequence reported. The molecular weight of the proC monomer is 28,112.     

Complementation of an Escherichia coli proC mutation by a gene cloned from Treponema pallidum.

Little is known concerning the biosynthetic and metabolic capabilities of the syphilis agent, Treponema pallidum, because of the inability to cultivate continuously the organism in vitro. To circumvent the problem of cultivation, researchers have used recombinant DNA technology to express treponemal protein antigens in Escherichia coli. However, with a few notable exceptions, the specific cellular roles of these cloned treponemal proteins have not been determined. In this study, a cosmid library of T. pallidum genomic DNA was constructed and amplified by repackaging infective lambda bacteriophage particles in vivo. Recombinant clones capable of complementing a null mutation in the E. coli proC gene encoding 1-pyrroline-5-carboxylate (P5C) reductase (EC 1.5.1.2) were subsequently identified. The complementing activity was eventually localized to a 2.3-kilobase BglII-HindIII fragment that hybridized to the same-size fragment of a BglII-HindIII digest of T. pallidum DNA. Two proteins of 41 and 27 kilodaltons (kDa) were encoded by this fragment, as determined by maxicell analysis. Although only the 41-kDa protein could be specifically precipitated by experimental syphilitic rabbit antisera, it was the 27-kDa protein that was responsible for the proC-complementing activity. The recombinant P5C reductase differed from the native E. coli enzyme by a number of biochemical properties. The cloning of a T. pallidum gene encoding P5C reductase strongly suggests that this pathogen has the ability to synthesize proline and possibly other amino acids.     

Development of a markerless genetic exchange method for Methanosarcina acetivorans C2A and its use in construction of new genetic tools for methanogenic archaea

A new genetic technique for constructing mutants of Methanosarcina acetivorans C2A by using hpt as a counterselectable marker was developed. Mutants with lesions in the hpt gene, encoding hypoxanthine phosphoribosyltransferase, were shown to be >35-fold more resistant to the toxic base analog 8-aza-2,6-diaminopurine (8ADP) than was the wild type. Reintroduction of the hpt gene into a Delta hpt host restored 8ADP sensitivity and provided the basis for a two-step strategy involving plasmid integration and excision for recombination of mutant alleles onto the M. acetivorans chromosome. We have designated this method markerless exchange because, although selectable markers are used during the process, they are removed in the final mutants. Thus, the method can be repeated many times in the same cell line. The method was validated by construction of Delta proC Delta hpt mutants, which were recovered at a frequency of 22%. Additionally, a Methanosarcina-Escherichia shuttle vector, encoding the Escherichia coli proC gene as a new selectable marker, was constructed for use in proC hosts. Finally, the markerless exchange method was used to recombine a series of uidA reporter gene fusions into the M. acetivorans proC locus. In vitro assay of beta-glucuronidase activity in extracts of these recombinants demonstrated, for the first time, the utility of uidA as a reporter gene in Methanosarcina: A >5,000-fold range of promoter activities could be measured by using uidA: the methyl-coenzyme M reductase operon fusion displayed approximately 300-fold-higher activity than did the serC gene fusion, which in turn had 16-fold-higher activity than did a fusion to the unknown orf2 gene.     

Identification of a new class of 5'-adenylylsulfate (APS) reductases from sulfate-assimilating bacteria

A gene was cloned from Burkholderia cepacia DBO1 that is homologous with Escherichia coli cysH encoding 3'-phosphoadenylylsulfate (PAPS) reductase. The B. cepacia gene is the most recent addition to a growing list of cysH homologs from a diverse group of sulfate-assimilating bacteria whose products show greater homology to plant 5'-adenylylsulfate (APS) reductase than they do to E. coli CysH. The evidence reported here shows that the cysH from one of the species, Pseudomonas aeruginosa, encodes APS reductase. It is able to complement an E. coli cysH mutant and a cysC mutant, indicating that the enzyme is able to bypass PAPS, synthesized by the cysC product. Insertional knockout mutation of P. aeruginosa cysH produced cysteine auxotrophy, indicating its role in sulfate assimilation. Purified P. aeruginosa CysH expressed as a His-tagged recombinant protein is able to reduce APS, but not PAPS. The enzyme has a specific activity of 5.8 micromol. min(-1). mg of protein(-1) at pH 8.5 and 30 degrees C with thioredoxin supplied as an electron donor. APS reductase activity was detected in several bacterial species from which the novel type of cysH has been cloned, indicating that this enzyme may be widespread. Although an APS reductase from dissimilatory sulfate-reducing bacteria is known, it shows no structural or sequence homology with the assimilatory-type APS reductase reported here. The results suggest that the dissimilatory and assimilatory APS reductases evolved convergently.     

Oxidation of 3,4-dehydro-D-proline and other D-amino acid analogues by D-alanine dehydrogenase from Escherichia coli

3,4-Dehydro-DL-proline is a toxic analogue of L-proline which has been useful in studying the uptake and metabolism of this key amino acid. When membrane fractions from Escherichia coli strain UMM5 (putA1::Tn5 proC24) lacking both L-proline dehydrogenase and L-Delta(1)-pyrroline-5-carboxylate reductase were incubated with 3,4-dehydro-DL-proline, pyrrole-2-carboxylate was formed. There was no enzyme activity with 3,4-dehydro-L-proline, but activity was restored after racemization of the substrate. Oxidation of 3,4-dehydro-DL-proline by membrane fractions from strain UMM5 was induced by growth in minimal medium containing D- or L-alanine, had a pH optimum of 9, and was competitively inhibited by D-alanine. An E. coli strain with no D-alanine dehydrogenase activity due to the dadA237 mutation was unable to oxidize either 3,4-dehydro-D-proline or D-alanine, as were spontaneous Dad(-) mutants of E. coli strain UMM5. Membrane fractions containing D-alanine dehydrogenase also catalyzed the oxidation of D-2-aminobutyrate, D-norvaline, D-norleucine, cis-4-hydroxy-D-proline, and DL-ethionine. These results indicate that d-alanine dehydrogenase is responsible for the residual 3,4-dehydro-DL-proline oxidation activity in putA proC mutants of E. coli and provide further evidence that this enzyme plays a general role in the metabolism of D-amino acids and their analogues.     

Microbial reduction of technetium by Escherichia coli and Desulfovibrio desulfuricans: enhancement via the use of high-activity strains and effect of process parameters

Escherichia coli and Desulfovibrio desulfuricans reduce Tc(VII) (TcO(4)(-)) with formate or hydrogen as electron donors. The reaction is catalyzed by the hydrogenase component of the formate hydrogenlyase complex (FHL) of E. coli and is associated with a periplasmic hydrogenase activity in D. desulfuricans. Tc(VII) reduction in E. coli by H(2) and formate was either inhibited or repressed by 10 mM nitrate. By contrast, Tc(VII) reduction catalyzed by D. desulfuricans was less sensitive to nitrate when formate was the electron donor, and unaffected by 10 mM or 100 mM nitrate when H(2) was the electron donor. The optimum pH for Tc(VII) reduction by both organisms was 5.5 and the optimum temperature was 40 degrees C and 20 degrees C for E. coli and D. desulfuricans, respectively. Both strains had an apparent K(m) for Tc(VII) of 0.5 mM, but Tc(VII) was removed from a solution of 300 nM TcO(4)(-) within 30 h by D. desulfuricans at the expense of H(2). The greater bioprocess potential of D. desulfuricans was shown also by the K(s) for formate (>25 mM and 0.5 mM for E. coli and D. desulfuricans, respectively), attributable to the more accessible, periplasmic localization of the enzyme in the latter. The relative rates of Tc(VII) reduction for E. coli and D. desulfuricans (with H(2)) were 12.5 and 800 micromol Tc(VII) reduced/g biomass/h, but the use of an E. coli HycA mutant (which upregulates FHL activities by approx. 50%) had a similarly enhancing effect on the rate of Tc reduction. The more rapid reduction of Tc(VII) by D. desulfuricans compared with the E. coli strains was also shown using cells immobilized in a hollow-fiber reactor, in which the flow residence times sustaining steady-state removal of 80% of the radionuclide were 24.3 h for the wild-type E. coli, 4.25 h for the upregulated mutant, and 1.5 h for D. desulfuricans.     

Mutations in the Corynebacterium glutamicum proline biosynthetic pathway: a natural bypass of th proA step.

Two chromosomal loci containing the Corynebacterium glutamicum ATCC 17965 proB and proC genes were isolated by complementation of Escherichia coli proB and proC auxotrophic mutants. Together with a proA gene described earlier, these new genes describe the major C. glutamicum proline biosynthetic pathway. The proB and proA genes, closely linked in most bacteria, are in C. glutamicum separated by a 304-amino-acid open reading frame (unk) whose predicted sequence resembles that of the 2-hydroxy acid dehydrogenases. C. glutamicum mutants that carry null alleles of proB, proA, and proC were constructed or isolated from mutagenized cultures. Single proC mutants are auxotrophic for proline and secrete delta1-pyrroline-5-carboxylate, which are the expected phenotypes of bacterial proC mutants. However, the phenotypes or proB and proA mutants are unexpected. A proB mutant has a pleiotropic phenotype, being both proline auxotrophic and affected in cell morphology. Null proA alleles still grow slowly under proline starvation, which suggests that a proA-independent bypass of this metabolic step exists in C. glutamicum. Since proA mutants are complemented by a plasmid that contains the wild-type asd gene of C. glutamicum, the asd gene may play a role in this bypass.     

NADH-细胞色素b5还原酶在大肠杆菌中的表达及其产物的纯化

为了探讨 NADH-细胞色素 b5还原酶基因突变引起遗传性高铁血红蛋白血症的分子病理机制 ,研究突变型 ( b5R)蛋白结构和功能的关系 ,用基因重组技术将野生型和突变型 ( C2 0 3Y) b5Rc DNA克隆于 p GEX- 2 T载体 ,在大肠杆菌 BL2 1中诱导表达 .Western印迹鉴定所表达的蛋白为GST- b5R融合蛋白 .应用谷胱甘肽 - Sepharose 4B亲和层析 ,还原型谷胱甘肽洗脱得到纯化的GST- b5R和 GST- b5RC2 0 3Y融合蛋白 .比较 GST- b5R和 GST- b5RC2 0 3Y酶活性及稳定性 ,发现野生型和突变型的酶活性基本相同 .但与野生型酶相比 ,突变型酶对热的稳定性较差 ,对胰蛋白酶更加敏感 .结果提示 ,C2 0 3Y突变可引起蛋白质二级结构改变而导致酶的稳定性下降 .     

Mapping of a new genetic locus responsible for ampicillin resistance in Escherichia coli.

The ampicillin resistance locus of three different ampicillin-resistant, temperature-sensitive Escherichia coli mutants was mapped between proC and purE and does not correspond to any of the known genes in this region. The mutant gene responsible for the temperature sensitivity and consequent morphological changes in each mutant strain was not located in the same 5-min region, even though the two mutants characteristics co-reverted at a very high frequency.     

Structural requirements of the unique disulphide bond and the proline-rich motif within the alpha4-alpha5 loop for larvicidal activity of the Bacillus thuringiensis Cry4Aa delta-endotoxin

Both the disulphide bond (Cys192-Cys199) and the proline-rich motif (Pro193ProAsnPro196) in the long loop connecting the alpha4-alpha5 transmembrane hairpin of the Cry4Aa mosquito-larvicidal protein have been found to be unique among the Bacillus thuringiensis Cry delta-endotoxins. In this study, their structural requirements for larvicidal activity of the Cry4Aa toxin were investigated. C192A and C199A mutant toxins were initially generated and over-expressed in Escherichia coli cells as 130-kDa protoxins at levels comparable to that of the wild-type toxin. When their activities against Aedes aegypti larvae were determined, Escherichia coli cells expressing each mutant toxin retained the high-level toxicity. Further mutagenic analysis of the PPNP motif revealed that an almost complete loss in larvicidal activity was observed for the C199A/P193A double mutant, whereas a small reduction in toxicity was shown for the C199A/P194A and C199A/P196A mutants. Increasing the flexibility of the alpha4-alpha5 loop through C199A/P193G, C199A/P194G/P196A, C199A/P194A/P196G, and C199A/P194G/P196G mutations significantly decreased the larvicidal activity. Similar to the wild-type protoxin, all mutant toxins were structurally stable upon solubilisation and trypsin activation in carbonate buffer, pH 9.0. These findings are the first biological evidence for a structural function in larvicidal activity of the unique disulphide bridge as well as the proline-rich motif within the alpha4-alpha5 loop of the Cry4Aa toxin.     

Ultraviolet Sensitivity Gene of Escherichia coli B

The ultraviolet sensitivity gene of Escherichia coli B was introduced into a K-12 recipient by transduction with phage P1. The uvs gene of E. coli B is cotransducible with the proC locus of K-12, is closely linked to tsx, is not linked to lacZ, and only rarely to purE. The transductants are mucoid, filamentous on irradiation, and show plating-medium response. The order of markers is lacZ proC tsx uvs purE.     

The interaction of 5'-adenylylsulfate reductase from Pseudomonas aeruginosa with its substrates

APS reductase from Pseudomonas aeruginosa has been shown to form a disulfide-linked adduct with mono-cysteine variants of Escherichia coli thioredoxin and Chlamydomonas reinhardtii thioredoxin h1. These adducts presumably represent trapped versions of the intermediates formed during the catalytic cycle of this thioredoxin-dependent enzyme. The oxidation-reduction midpoint potential of the disulfide bond in the P. aeruginosa APS reductase/C. reinhardtii thioredoxin h1 adduct is -280 mV. Site-directed mutagenesis and mass spectrometry have identified Cys256 as the P. aeruginosa APS reductase residue that forms a disulfide bond with Cys36 of C. reinhardtii TRX h1 and Cys32 of E. coli thioredoxin in these adducts. Spectral perturbation measurements indicate that P. aeruginosa APS reductase can also form a non-covalent complex with E. coli thioredoxin and with C. reinhardtii thioredoxin h1. Perturbation of the resonance Raman and visible-region absorbance spectra of the APS reductase [4Fe-4S] center by either APS or the competitive inhibitor 5'-AMP indicates that both the substrate and product bind in close proximity to the cluster. These results have been interpreted in terms of a scheme in which one of the redox-active cysteine residues serves as the initial reductant for APS bound at or in close proximity to the [4Fe-4S] cluster.     

The Bradyrhizobium japonicum proline biosynthesis gene proC is essential for symbiosis

Plant host-derived proline is proposed to serve as an energy source for rhizobia in the rhizosphere and in symbiotic root nodules. The Bradyrhizobium japonicum proC gene was isolated, and a proC mutant strain that behaved as a strict proline auxotroph in culture was constructed. The proC strain elicited undeveloped nodules on soybeans that lacked nitrogen fixation activity and plant hemoglobin. We conclude that the proC gene is essential for symbiosis and suggest that the mutant does not obtain an exogenous supply of proline in association with soybeans sufficient to satisfy its auxotrophy.     

盐胁迫下小麦苗叶片吡咯-5-羧酸还原酶活性和游离脯氨酸积累

受NaCl、KCl或MsCl_2胁迫的小麦幼苗,当外部溶液的渗透势由—160 kPa下降到—900 kPa时,叶片吡咯—5—羧酸还原酶(PSC)活性增高;渗透势由—900 kPa降低到—1500kPa,酶活性下降;胁迫 1d和 2d的幼苗,还原酶活性显著增加;3～6d酶活性无大变化。游离脯氨酸含量随溶液渗透势下降和培养时间的延长而提高。胁迫解除后酶活性和脯氨酸含量均降低。受NcCl胁迫义在ABA影响下的幼苗,P5C还原酶活性和脯氨酸含量高于仅受NaCl胁迫的幼苗。     

Pantothenate production in Escherichia coli K12 by enhanced expression of the panE gene encoding ketopantoate reductase.

Using gene replacement and transposon Tn5 mutagenesis, an Escherichia coli ilvC panE double mutant completely lacking ketopantoate reductase activity was isolated. This E. coli double mutant was employed to isolate the E. coli panE gene by genetic complementation. The E. coli panE gene is characterized by a 912 bp coding region, which specifies a protein of 303 amino acids with a deduced molecular mass of 33.8 kD. A panE expression plasmid carrying the panE gene under the control of the tac promotor was constructed. Introduction of the panE expression plasmid into E. coli resulted in a threefold increase in ketopantoate reductase activity. It was also shown that the enhanced panE expression in E. coli K12 led to 3.5-fold increase in pantothenate excretion. Pantothenate excretion could even be more enhanced when the growth medium was supplemented with ketopantoate.