The three-dimensional structure of Escherichia coli porphobilinogen deaminase at 1.76-Å resolution

Porphobilinogen deaminase (PBGD) catalyses the polymerization of four molecules of porphobilinogen to form the 1-hydroxymethylbilane, preuroporphyrinogen, a key intermediate in the biosynthesis of tetrapyrroles. The three-dimensional structure of wild-type PBGD from Escherichia coli has been determined by multiple isomorphous replacement and refined to a crystallographic R-factor of 0.188 at 1.76 Å resolution. The polypeptide chain of PBGD is folded into three α/β domains. Domains 1 and 2 have a similar overall topology, based on a five-stranded, mixed β-sheet. These two domains, which are linked by two hinge segments but otherwise make few direct interactions, form an extensive active site cleft at their interface. Domain 3, an open-faced, anti-parallel sheet of three strands, interacts approximately equally with the other two domains. The dipyrromethane cofactor is covalently attached to a cysteine side-chain borne on a flexible loop of domain 3. The cofactor serves as a primer for the assembly of the tetrapyrrole product and is held within the active site cleft by hydrogen-bonds and salt-bridges that are formed between its acetate and propionate side-groups and the polypeptide chain. The structure of a variant of PBGD, in which the methionines have been replaced with selenomethionines, has also been determined. The cofactor, in the native and functional form of the enzyme, adopts a conformation in which the second pyrrole ring (C2) occupies an internal position in the active site cleft. On oxidation, however, this C2 ring of the cofactor adopts a more external position that may correspond approximately to the site of substrate binding and polypyrrole chain elongation. The side-chain of Asp84 hydrogen-bonds the hydrogen atoms of both cofactor pyrrole nitrogens and also potentially the hydrogen atom of the pyrrole nitrogen of the porphobilinogen molecule bound to the proposed substrate binding site. This group has a key catalytic role, possibly in stabilizing the positive charges that develop on the pyrrole nitrogens during the ring-coupling reactions. Possible mechanisms for the processive elongation of the polypyrrole chain involve: accommodation of the elongating chain within the active site cleft, coupled with shifts in the relative positions of domains 1 and 2 to carry the terminal ring into the appropriate position at the catalytic site; or sequential translocation of the elongating polypyrrole chain, attached to the cofactor on domain 3, through the active site cleft by the progressive movement of domain 3 with respect to domains 1 and 2. Other mechanisms are considered although the amino acid sequence comparisons between PBGDs from all species suggest they share the same three-dimensional structure and mechanism of activity. © 1996 Wiley-Liss, Inc.     

大肠杆菌代谢工程改造合成L-高丝氨酸及其衍生物研究进展

l-高丝氨酸及其衍生物(O-琥珀酰-l-高丝氨酸和O-乙酰-l-高丝氨酸)是生物合成l-甲硫氨酸的前体,同时也是合成多种C4化合物(异丁醇、g-丁内酯、1,4-丁二醇、2,4-二羟基丁酸等)和l-草铵膦等的平台化合物。因此,发酵法生产l-高丝氨酸及其衍生物成为近年内研究的热点。然而,利用生物法合成l-高丝氨酸及其衍生物仍存在一些不足之处,如发酵产量不高或糖酸转化率过低等。此外,对l-高丝氨酸及其衍生物合成的总体代谢和调控机制鲜有报道。本文综述了大肠杆菌代谢工程改造合成l-高丝氨酸及其衍生物O-琥珀酰-l-高丝氨酸和O-乙酰-l-高丝氨酸的研究进展,从底物摄取、关键节点碳流分配改造、辅酶NADPH的循环供应以及目标产物的外运输出等方面,系统分析了大肠杆菌全发酵法生产l-高丝氨酸及其衍生物的代谢途径及改造策略,为其后续代谢改造及生物法生产提供一定的研究思路。     

Development of a bacterial model for studying anthracycline-membrane interactions

Abstract Growth of wild-type Escherichia coli strain MRE600 was severely affected up to 9 h following treatment with the anthracycline doxorubicin (15 μM), however, after 9 h, the cells became resistant. The onset of resistance coincided with some changes in the relative proportions of total saturated, monounsaturated and cyclopropane fatty acids. The anionic lipid content in E. coli strain HDL11 is under lac control and synthesis can be induced by incubation with the lac inducer IPTG. HDL11, with low levels of anionic phospholipid, was unaffected by doxorubicin (100 μM) over 9 h, with only slight inhibition of growth seen over 24 h. When the anionic lipid content of HDL11 was increased, there was a slight increase in the efficacy of doxorubicin, providing evidence for a membrane-based step in doxorubicin action.     

Molecular cloning and expression of the glucose/xylose isomerase gene from Streptomyces sp. NCIM 2730 in Escherichia coli

Abstract A partial genomic library of Streptomyces sp. NCIM 2730 was constructed in Escherichia coli using pUC8 vector and screened for the presence of the d-glucose/xylose isomerase (GXI) gene using an 18-mer mixed oligonucleotide probe complementary to a highly conserved six-amino acid sequence of GXI from actinomycetes. Eight clones which hybridized with the radiolabelled oligoprobe showed the ability to complement xylose isomerase-defective E. coli mutants. The restriction map of the insert from one (pMSG27) of the eight GXI-positive clones showing detectable GXI activity was constructed. GXI-deficient strains of E. coli were able to utilize xylose as the sole carbon source for their growth upon transformation with pMSG27. E. coli JM105 (pMSG27) and E. coli JC1553 (pMSG27) were inducible by IPTG suggesting that the expression of the cloned gene was under the control of the lacZ promoter. Western blot analysis revealed that the cloned gene is expressed as a fusion protein of M _r 110. This is the first report of expression of a catalytically active GXI from Streptomyces in Escherichia coli .     

Enterotoxin-like activity produced by Campylobacter jejuni and Campylobacter coli isolated from patients and healthy controls in Algeria

Abstract In our earlier studies, hexammine ruthenium (III) chloride (HRC) was found to eliminate the pBR322 plasmid from E. coli HB101 with 100% frequency. However, this plasmid was not eliminated by other known curing agents, such as acridine orange, sodium dodecyl sulphate (SDS), rifampicin or temperature conditions. In our present communication, we report the sensitivity of a variant of plasmid pBR322 to curing agents which were not effective on the parent plasmid.     

Participation of hup gene product in ori2-dependent replication of fertility plasmid F

The fertility plasmid F'gal was not stably maintained in a hupA-hupB double mutant of Escherichia coli. Moreover, mini-F plasmids pFZY1, pFTC1 and pFTC2 were unable to transform the double mutant, though these plasmids efficiently transformed cells harboring a hupA or hupB single mutation. The composite plasmid pFHS1, which consists of the f5 DNA fragment of F plasmid and the whole DNA of a pSC101 derivative that carries a temperature-sensitive mutation for DNA replication, was not stably maintained in the hup double mutant at 42°C. These findings strongly suggest that HU protein is required for ori2-dependent replication of the F plasmid.     

Low affinity, antibody binding of an Escherichia coli-derived component

Abstract This investigation describes the detection of a component in Escherichia coli capable of binding a large proportion of human antibody variable domains including otherwise highly monospecific antibodies induced by an in vivo antibody response. This interaction is of low affinity, but cross-linking of IgG molecules by e.g. anti-immunoglobulin preparations, provides a sufficient degree of multivalency to promote a high avidity interaction. This binding which occurs both with κ and λ light chain-containing antibodies, appears to involve the variable region of human antibodies making it a superantigen-like activity. This is proposed based on the facts that: (i) different human antibodies of IgG1 isotype appear to bind to different extents suggesting that variable domain differences determine the binding activity; and (ii) addition of soluble antigen abrogates the interaction with the E. coli -derived molecule. Future studies of the nature and possible in vivo consequences of these interactions are warranted since any superantigen activity associated with this binding might affect the human immune response occurring as a consequence of E. coli infections.     

Monoclonal antibodies against pertussis toxin subunits

Abstract Twenty monoclonal antibodies (mAbs) reacting with cholera toxin (CT) of Vibrio cholerae strain 569B were characterized in cross-section and G_M1 ganglioside inhibition assays. MAbs were characterized by reaction with CT and Escherichia coli heat-labile porcine strain (LT_p) and human strain (LTh) enterotoxins, and by G_M1 ganglioside inhibition of mAb binding. Eight of 10 CT-A specific and 3 of 10 CT-B-specific mAbs cross-reacted with LTh and LTp. G_M1 ganglioside inhibited reactions of the CT-B cross-reacting antibodies. Results showed that these epitodes common to the B subunit of CT and LT are located in or near the G_M1 ganglioside binding region, and that the G_M1 ganglioside-binding region of LT differs from that of CT.     

Tn10 generated deletions of the ompB locus of Escherichia coli K12

Abstract We have isolated a set of Tn 10 -generated deletions starting from the distal end of the ompR envZ operon of Escherichia coli K12. Most of the deletions removed both ompR and envZ genes or ended in ompR . These deletions exhibited an OmpC⁻ OmpF⁻ phenotype. One deletion removed only part of envZ and the strain was phenotypically OmpC⁻ OmpF^+/−. This deletion of the distal part of envZ did not affect osmoregulation of ompC . However, ompF osmoregulation appeared reversed. High osmolarity in the growth medium resulted in production of OmpF close to the wild-type level.