A novel L-serine deaminase activity in Escherichia coli K-12.

We demonstrate here that Escherichia coli K-12 synthesizes two different L-serine deaminases (L-SD) catalyzing the nonoxidative deamination of L-serine to pyruvate, one coded for by the previously described sdaA gene and a second, hitherto undescribed enzyme which we call L-SD2. A strain carrying a null mutation in sdaA made no detectable L-SD in minimal medium, but had activity in Luria broth. We describe a mutation, sdaX, which affects the regulation of L-SD2 and permits its expression in minimal medium, and an insertion mutation, sdaB, which abolishes L-SD2 activity completely. Both mutations lie near 60.5 min on the E. coli genetic map. The two L-SD enzymes have similar enzyme parameters, and both require posttranslational activation.     

Metabolic engineering of Corynebacterium glutamicum for L-serine production

Although L-serine proceeds in just three steps from the glycolytic intermediate 3-phosphoglycerate, and as much as 8% of the carbon assimilated from glucose is directed via L-serine formation, previous attempts to obtain a strain producing L-serine from glucose have not been successful. We functionally identified the genes serC and serB from Corynebacterium glutamicum, coding for phosphoserine aminotransferase and phosphoserine phosphatase, respectively. The overexpression of these genes, together with the third biosynthetic serA gene, serA(delta197), encoding an L-serine-insensitive 3-phosphoglycerate dehydrogenase, yielded only traces of L-serine, as did the overexpression of these genes in a strain with the L-serine dehydratase gene sdaA deleted. However, reduced expression of the serine hydroxymethyltransferase gene glyA, in combination with the overexpression of serA(delta197), serC, and serB, resulted in a transient accumulation of up to 16 mM L-serine in the culture medium. When sdaA was also deleted, the resulting strain, C. glutamicum delta sdaA::pK18mobglyA'(pEC-T18mob2serA(delta197)CB), accumulated up to 86 mM L-serine with a maximal specific productivity of 1.2 mmol h(-1) g (dry weight)(-1). This illustrates a high rate of L-serine formation and also utilization in the C. glutamicum wild type. Therefore, metabolic engineering of L-serine production from glucose can be achieved only by addressing the apparent key position of this amino acid in the central metabolism.     

谷氨酸棒杆菌SYPS-062 L-丝氨酸脱水酶活性分析及其基因敲除对L-丝氨酸积累的影响

以谷氨酸棒杆菌(Corynebacterium glutamicum) SYPS-062基因组DNA为模板,扩增得到L-丝氨酸脱水酶(L-SerDH)的编码基因sdaA。将其克隆到表达载体pET-28a(+),并在E.coli BL21(DE3)中诱导表达,对纯化的L-SerDH进行了酶活测定,并与来自C.glutamicum ATCC13032的重组L-SerDH进行了比较,结果显示,两种不同菌株来源的重组L-SerDH降解L-丝氨酸的酶比活力差异并不显著。在此基础上敲除菌株SYPS-062 的sdaA基因,探讨该基因对C.glutamicum SYPS-062生长及产酸的影响。通过构建自杀型重组质粒pK18mobsacB-△sdaA,电击转入C.glutamicum SYPS-062中,以同源重组的方式获得了sdaA基因缺失突变株,并用PCR方法对突变株C.glutamicum SYPS-062△sdaA进行了验证。与出发菌株相比,突变菌株生长缓慢,单位菌体L-丝氨酸的产量(Y_P/X)提高了15.13%。     

Deficiency in l-serine deaminase results in abnormal growth and cell division of Escherichia coli K-12

The loss of the ability to deaminate l -serine severely impairs growth and cell division in Escherichia coli K-12. A strain from which the three genes ( sdaA , sdaB , tdcG ) coding for this organism's three l -serine deaminases had been deleted grows well in glucose minimal medium but, on subculture into minimal medium with glucose and casamino acids, it makes very large, abnormally shaped cells, many of which lyse. When inoculated into Luria-Bertani (LB) broth with or without glucose, it makes very long filaments. Provision of S-adenosylmethionine restores cell division in LB broth with glucose, and repairs much of the difficulty in growth in medium with casamino acids. We suggest that replication of E. coli is regulated by methylation, that an unusually high intracellular l -serine concentration, in the presence of other amino acids, starves the cell for S-adenosylmethionine and that it is the absence of S-adenosylmethionine and/or of C1-tetrahydrofolate derivatives that prevents normal cell division.     

Cometabolism of a nongrowth substrate: L-serine utilization by Corynebacterium glutamicum

Despite its key position in central metabolism, L-serine does not support the growth of Corynebacterium glutamicum. Nevertheless, during growth on glucose, L-serine is consumed at rates up to 19.4 +/- 4.0 nmol min(-1) (mg [dry weight])(-1), resulting in the complete consumption of 100 mM L-serine in the presence of 100 mM glucose and an increased growth yield of about 20%. Use of 13C-labeled L-serine and analysis of cellularly derived metabolites by nuclear magnetic resonance spectroscopy revealed that the carbon skeleton of L-serine is mainly converted to pyruvate-derived metabolites such as L-alanine. The sdaA gene was identified in the genome of C. glutamicum, and overexpression of sdaA resulted in (i) functional L-serine dehydratase (L-SerDH) activity, and therefore conversion of L-serine to pyruvate, and (ii) growth of the recombinant strain on L-serine as the single substrate. In contrast, deletion of sdaA decreased the L-serine cometabolism rate with glucose by 47% but still resulted in degradation of L-serine to pyruvate. Cystathionine beta-lyase was additionally found to convert L-serine to pyruvate, and the respective metC gene was induced 2.4-fold under high internal L-serine concentrations. Upon sdaA overexpression, the growth rate on glucose is reduced 36% from that of the wild type, illustrating that even with glucose as a single substrate, intracellular L-serine conversion to pyruvate might occur, although probably the weak affinity of L-SerDH (apparent Km, 11 mM) prevents substantial L-serine degradation.     

Characterization,modification, and overexpression of 3-phosphoglycerate dehydrogenase in Corynebacterium glutamicum for enhancing l-serine production

The direct fermentative production of l-serine from renewable biomass using Corynebacterium glutamicum is attracting increasing attention. In this study, wild-type C. glutamicum SYPS-062 produced up to 6.65 ± 0.23 g/L l-serine; to further improve l-serine production, the serA gene was cloned, and the C-terminal domain of 3-phosphoglycerate dehydrogenase (PGDH) from this strain was truncated. When expressed in Escherichia coli, the resultant mutein SerAΔ197 showed a specific PGDH activity of 1.092 ± 0.05 U/mg protein, representing a decrease of 25.87 % from that encoded by serA, and was no longer sensitive to high concentrations of l-serine. When serA Δ591 was overexpressed in C. glutamicum SYPS-062, the activity of PGDH in C. glutamicum pJC1-tac-serA Δ591 increased by 47.72 %, and the resultant strain C. glutamicum pJC1-tac-serA Δ591 could accumulate 7.69 ± 0.22 g/L l-serine. Furthermore, when serA Δ591 was overexpressed in C. glutamicum SYPS-062ΔsdaA, the resultant strain could accumulate 8.84 ± 0.23 g/L l-serine at 102 h, and the yield of l-serine on cells (Y p/x) improved by 60 % when compared with that noted in the control. These results demonstrate that l-serine production in C. glutamicum SYPS-062 could be improved by overexpressing a C-terminal truncation of PGDH in combination with other genetic modifications.     

L-serine degradation in Escherichia coli K-12: cloning and sequencing of the sdaA gene.

A new mutant of Escherichia coli K-12 unable to grow with L-serine, glycine, and L-leucine has been isolated by lambda plac Mu insertion and shown to be deficient in L-serine deaminase activity. The corresponding gene, sdaA, has been cloned from a prototrophic strain, and the clone has been characterized and sequenced. The evidence is consistent with the hypothesis that sdaA is the structural gene for L-serine deaminase. However, other possibilities are also considered. No significant homology with previously reported DNA or protein sequences was detected.     

具有高谷胱甘肽合成活性重组大肠杆菌的构建及合成反应过程

以野生型大肠杆菌E.coliⅡ为宿主细胞,转化带有编码谷胱甘肽合成酶系的基因gshⅠ和gshⅡ的质粒pGH501,获得了一株谷胱甘肽合成活性、质粒稳定性和传代稳定性俱佳,并且能够重复使用的重组大肠杆菌E.coliⅡ\|1。该菌株经过甲苯处理后,能够在胞外积累4g/L左右的谷胱甘肽(GSH)。在合成反应体系中,提高L谷氨酸浓度可促进GSH合成,但L半胱氨酸浓度增大到20mmol/L后会抑制GSH的合成。根据GSH合成反应中能量辅因子的变化情况,提出E.coliⅡ\|1细胞控制的GSH合成反应机理：由谷胱甘肽合成酶(GSHⅡ)控制的第二步反应的能量供体是ADP而非ATP,该反应是整个GSH合成反应的限速步骤,高浓度ADP可能会抑制GSHⅡ的活性。在GSH合成反应体系中添加100mmol/L的L丝氨酸-硼酸钾混合物,可以有效地防止GSH的进一步降解,反应3 h后,GSH产量达到230mmol/L(约71g/L)。     

D-3-Phosphoglycerate dehydrogenase from Mycobacterium tuberculosis is a link between the Escherichia coli and mammalian enzymes

D-3-Phosphoglycerate dehydrogenase (PGDH) from Mycobacterium tuberculosis has been isolated to homogeneity and displays an unusual relationship to the Escherichia coli and mammalian enzymes. In almost all aspects investigated, the M. tuberculosis enzyme shares the characteristics of the mammalian PGDHs. These include an extended C-terminal motif, substrate inhibition kinetics, dependence of activity levels and stability on ionic strength, and the inability to utilize alpha-ketoglutarate as a substrate. The unique property that the M. tuberculosis enzyme shares with E. coli PGDH that it is very sensitive to inhibition by L-serine, with an I(0.5) = 30 microm. The mammalian enzymes are not inhibited by L-serine. In addition, the cooperativity of serine inhibition appears to be modulated by chloride ion, becoming positively cooperative in its presence. This is modulated by the gain of cooperativity in serine binding for the first two effector sites. The basis for the chloride modulation of cooperativity is not known, but the sensitivity to serine inhibition can be explained in terms of certain amino acid residues in critical areas of the structures. The differential sensitivity to serine inhibition by M. tuberculosis and human PGDH may open up interesting possibilities in the treatment of multidrug-resistant tuberculosis.     

3-Phosphoglycerate dehydrogenase from Corynebacterium glutamicum: the C-terminal domain is not essential for activity but is required for inhibition by L-serine

The serA gene of Corynebacterium glutamicum coding for 3-phosphoglycerate dehydrogenase (PGDH) was isolated and functionally characterized. It encodes a polypeptide of 530 aminoacyl residues (aa), which is substantially longer than the corresponding Escherichia coli polypeptide of 410 aa. The difference is largely due to an additional stretch of aa in the carboxy- (C)-terminal part of the polypeptide. Overexpression of serA in C. glutamicum results in a 16-fold increase in specific PGDH activity to 2.1 U/mg protein, with activity being inhibited by high concentrations of L-serine. A set of muteins that were progressively truncated at the C-terminal end was constructed. When overexpressed, mutein SerADelta197 showed a specific PGDH dehydrogenase activity of 1.3 U/mg protein, with the activity no longer being sensitive to L-serine. Gel filtration experiments showed that wild type PGDH is a homotetramer, whereas mutein SerADelta197 constitutes a dimer. Thus, the specific regulatory features of C. glutamicum PGDH are due to the C-terminal part of the polypeptide, which can be deleted with almost no effect on the catalytic activity of the enzyme.     

大肠杆菌PGDH末端缺失突变体的构建及抗反馈抑制效应分析

为了获得具有抗反馈抑制性质的大肠杆菌磷酸甘油酸脱氢酶（PGDH, d-3-phosphoglycerate dehydrogenase, EC 1.1.1.95）,通过对其碱基序列和蛋白质结构分析,用PCR突变法构建突变酶M1（缺失第410位氨基酸）、M2（缺失407~410位氨基酸）、M3（缺失337~410位氨基酸）。M0(野生型)及各突变型基因与pET22b(+)载体连接后,表达融合蛋白。在非变性条件下,由NTA-Ni镍离子螯合亲和层析柱纯化野生型和突变体的酶蛋白。酶活性测定结果表明,M1、M2蛋白酶均保持了原有的野生型磷酸甘油酸脱氢酶活性,且部分解除了终产物L-丝氨酸的反馈抑制作用;M3蛋白酶完全解除了终产物的反馈抑制作用,但酶本身的催化活性略有降低（为野生型的83%）。M0、M1、M2菌株PGDH与L-丝氨酸结合的Ki值分别约为7 μmol/L、20 μmol/L、50 μmol/L,说明该酶C-末端1~4个氨基酸残基对L-丝氨酸和调控区的结合有重要影响。     

Escherichia coli L-serine deaminase requires a [4Fe-4S] cluster in catalysis

L-Serine deaminases catalyze the deamination of L-serine, producing pyruvate and ammonia. Two families of these proteins have been described and are delineated by the cofactor that each employs in catalysis. These are the pyridoxal 5'-phosphate-dependent deaminases and the deaminases that are activated in vitro by iron and dithiothreitol. In contrast to the enzymes that employ pyridoxal 5'-phosphate, detailed physical and mechanistic characterization of the iron-dependent deaminases is limited, primarily because of their extreme instability. We report here the characterization of L-serine deaminase from Escherichia coli, which is the product of the sdaA gene. When purified anaerobically, the isolated protein contains 1.86 +/- 0.46 eq of iron and 0.670 +/- 0.019 eq of sulfide per polypeptide and displays a UV-visible spectrum that is consistent with a [4Fe-4S] cluster. Reconstitution of the protein with iron and sulfide generates considerably more of the cluster, and treatment of the reconstituted protein with dithionite gives rise to an axial EPR spectrum, displaying g axially = 2.03 and g radially = 1.93. M?ssbauer spectra of the (57)Fe-reconstituted protein reveal that the majority of the iron is in the form of [4Fe-4S](2+) clusters, as evidenced by the typical M?ssbauer parameters-isomer shift, delta = 0.47 mm/s, quadrupole splitting of Delta E(Q) = 1.14 mm/s, and a diamagnetic (S = 0) ground state. Treatment of the dithionite-reduced protein with L-serine results in a slight broadening of the feature at g = 2.03 in the EPR spectrum of the protein, and a dramatic loss in signal intensity, suggesting that the amino acid interacts directly with the cluster.     

前列腺素脱氢酶在大肠杆菌中的表达纯化及鉴定

15-羟基前列腺素脱氢酶（PGDH）属于抑癌基因,在多种肿瘤中表达缺失,在肿瘤的发生发展中起着重要作用。提取人正常大肠黏膜组织总RNA,利用RT-PCR方法扩增得到PGDH基因的编码序列,克隆入原核表达载体pBV220,测序鉴定正确后转化E.coli DH5α,经温控诱导表达,表达产物进行SDS-PAGE和Western blot,证实为相对分子质量约为29000的PGDH-His6蛋白,表达产物以包涵体形式存在,3h诱导表达量最高,约占菌体总蛋白的30%。经Ni2+配体亲和层析纯化得到纯度大于95%的目的蛋白。重组PGDH简单复性后具有一定的生物活性,约为3.7×104U/mg,为下一步研究其在肿瘤中的作用奠定了基础。     

Uptake of phosphate and its effect on phenylalanine ammonia-lyase activity and cinnamoyl putrescine accumulation in cell suspension cultures of Nicotiana tabacum

The influence of phosphate on the medium-induced formation of cinnamoyl putrescines in cell cultures of Nicotiana tabcum was investigated. Phosphate added to a phosphate-free production medium was completely accumulated in the cells within 24h after inoculation at initial concentrations up to 2 mM. At higher concentrations phosphate was partly accumulated with an intracellular saturation at approx. 0.65 mmol/g dr. wt. equivalent to approx. 45 mM intracellular concentration. Enhanced activities of phenylalanine ammonialyase and increased product levels of cinnamoyl putrescines, induced by cell transfer into phosphatefree medium were suppressed similarly at initial phosphate concentrations of 0.02–0.5 mM. At the same time growth was stimulated.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - fr. wt. fresh weight - dr. wt. dry weight - MS-medium Murashige-Skoog-medium (Murashige and Skoog 1962)     

Identification of amino acid residues contributing to the mechanism of cooperativity in Escherichia coli D-3-phosphoglycerate dehydrogenase

L-Serine inhibits the catalytic activity of Escherichia coli D-3-phosphoglycerate dehydrogenase (PGDH) by binding to its regulatory domain. This domain is a member of the ACT domain family of regulatory domains that are modulated by small molecules. A comparison of the phi and psi torsional angle differences between the crystal structures of PGDH solved in the presence and in the absence of L-serine demonstrated a clustering of significant angle deviations in the regulatory domain. A similar clustering was not observed in either of the other two structural domains of PGDH. In addition, significant differences were also observed at the active site and in the Trp-139 loop. To determine if these residues were functionally significant and not just due to other factors such as crystal packing, mutagenic analysis of these residues was performed. Not unexpectedly, this analysis showed that residues that affected the kcat/Km were grouped around the active site and those that affected the serine sensitivity were grouped in the regulatory domain. However, more significantly, residues that affected the cooperativity of inhibition of activity were identified at both locations. These latter residues represent structural elements that participate in both the initial and the ultimate events of the transfer of cooperative behavior from the regulatory domain to the active site. As such, their identification will assist in the elucidation of the pathway of cooperative interaction in this enzyme as well as in the elucidation of the regulatory mechanism of the ACT domain in general.     

The contribution of adjacent subunits to the active sites of D-3-phosphoglycerate dehydrogenase

D-3-Phosphoglycerate dehydrogenase (PGDH) from Escherichia coli is allosterically inhibited by L-serine, the end product of its metabolic pathway. Previous results have shown that inhibition by serine has a large effect on Vmax and only a small or negligible effect on Km. PGDH is thus classified as a V-type allosteric enzyme. In this study, the active site of PGDH has been studied by site-directed mutagenesis to assess the role of certain residues in substrate binding and catalysis. These consist of a group of cationic residues (Arg-240, Arg-60, Arg-62, Lys-39, and Lys-141') that potentially form an electrostatic environment for the binding of the negatively charged substrate, as well as the only tryptophan residue found in PGDH and which fits into a hydrophobic pocket immediately adjacent to the active site histidine residue. Interestingly, Trp-139' and Lys-141' are part of the polypeptide chain of the subunit that is adjacent to the active site. The results of mutating these residues show that Arg-240, Arg-60, Arg-62, and Lys-141' play distinct roles in the binding of the substrate to the active site. Mutants of Trp-139' show that this residue may play a role in stabilizing the catalytic center of the enzyme. Furthermore, these mutants appear to have a significant effect on the cooperativity of serine inhibition and suggest a possible role for Trp-139' in the cooperative interactions between subunits.     

The production of L-serine with a methylotrophic microorganism using the L-serine pathway and coupling with an L-tryptophan-producing process

The cultural conditions for the production of enhanced formation of L-serine (up to 7 g/L) are described with the methylotrophic bacterium Pseudomonas 3 ab (DSM 672). The batch process is divided into three parts: (1) the biomass production phase, (2) substrate limitation period, and (3) L-serine accumulation phase. The initial specific production rate of q(p) = 0.1 g L-serine/g dry wt/h is based on the inhibition of the L-serine pathway. This is accomplished by high precursor concentrations (glycine) and a pH shift to pH 8.5. The enzymatic background is discussed. Furthermore, a coupling of the L-serine process with a L-tryptophan-producing process is demonstrated.     

L-serine production by a methionine-auxotrophic mutant of methylotrophic Pseudomonas

A methionine-auxotropic mutant deficient in homocysteine transmethylation activity was induced from a methylotrophic L-serine-producing derivatives of Pseudomonas MS31. This mutant grown with limited L-methionine had more than 1.7-fold higher serine hydroxymethyltransferase (SHMT) activity than its parent strain. The elevated SHMT activity significantly contributed to the improvement of L-serine accumulation from glycine and methanol. Under the optimum conditions, this mutant accumulated up to 23.9 mg/ml of L-serine. The yield coefficient L-serine from consumed glycine was 89% (mol/mol). The maximum conversion rate of added glycine (19 mg/ml) to L-serine was 77% (mol/mol).     

Characterization of short-chain dehydrogenase/reductase homologues of Escherichia coli (YdfG) and Saccharomyces cerevisiae (YMR226C)

Short-chain dehydrogenase/reductase homologues from Escherichia coli (YdfG) and Saccharomyces cerevisiae (YMR226C) show high sequence similarity to serine dehydrogenase from Agrobacterium tumefaciens. We cloned each gene encoding YdfG and YMR226C into E. coli JM109 and purified them to homogeneity from the E. coli clones. YdfG and YMR226C consist of four identical subunits with a molecular mass of 27 and 29 kDa, respectively. Both enzymes require NADP(+) as a coenzyme and use L-serine as a substrate. Both enzymes show maximum activity at about pH 8.5 for the oxidation of L-serine. They also catalyze the oxidation of D-serine, L-allo-threonine, D-threonine, 3-hydroxyisobutyrate, and 3-hydroxybutyrate. The k(cat)/K(m) values of YdfG for L-serine, D-serine, L-allo-threonine, D-threonine, L-3-hydroxyisobutyrate, and D-3-hydroxyisobutyrate are 105, 29, 199, 109, 67, and 62 M(-1) s(-1), and those of YMR226C are 116, 110, 14600, 7540, 558, and 151 M(-1) s(-1), respectively. Thus, YdfG and YMR226C are NADP(+)-dependent dehydrogenases acting on 3-hydroxy acids with a three- or four-carbon chain, and L-allo-threonine is the best substrate for both enzymes.     

Pentose phosphate pathway flux analysis for glycopeptide antibiotic vancomycin production during glucose-limited cultivation of Amycolatopsis orientalis

In vivo pentose phosphate pathway (PPP) enzymes such as glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), and transaldolase (TAL) activities as well as ATP- and ADP-level variations of Amycolatopsis orientalis were investigated with respect to glucose concentration and incubation period. G6PDH, 6PGDH, and TAL activities of A. orientalis reached maximum levels at 48 hr for all glucose concentrations used, after which the levels began to decline. G6PDH, 6PGDH, and TAL activities showed positive correlation with the glucose concentration up to 15 g/L, while further increases had an opposite effect. Intracellular ATP level showed a positive correlation with glucose concentrations, while ADP level increased up to 15 g/L. ATP concentration of A. orientalis increased rapidly at 48 hr of incubation, as was the case also for G6PDH, 6PGDH, and TAL activities, although the incubation period corresponding to maximum values of ADP shifted to 60 hr. Production of the glycopeptide antibiotic vancomycin increased with the increases in glucose concentrations up to 15 g/L, by showing coherence in the rates of oxidative and nonoxidative parts of the PPP.