Methionine sulfoxide reductase from the hyperthermophilic archaeon Thermococcus kodakaraensis, an enzyme designed to function at suboptimal growth temperatures

Methionine sulfoxide reductase (Msr) catalyzes the thioredoxin-dependent reduction and repair of methionine sulfoxide (MetO). Although Msr genes are not present in most hyperthermophile genomes, an Msr homolog encoding an MsrA-MsrB fusion protein (MsrAB(Tk)) was present on the genome of the hyperthermophilic archaeon Thermococcus kodakaraensis. Recombinant proteins corresponding to MsrAB(Tk) and the individual domains (MsrA(Tk) and MsrB(Tk)) were produced, purified, and biochemically examined. MsrA(Tk) and MsrB(Tk) displayed strict substrate selectivity for Met-S-O and Met-R-O, respectively. MsrAB(Tk), and in particular the MsrB domain of this protein, displayed an intriguing behavior for an enzyme from a hyperthermophile. While MsrAB(Tk) was relatively stable at temperatures up to 80 degrees C (with a half-life of approximately 30 min at 80 degrees C), a 75% decrease in activity was observed after 2.5 min at 85 degrees C, the optimal growth temperature of this archaeon. Moreover, maximal levels of MsrB activity of MsrAB(Tk) were observed at the strikingly low temperature of 30 degrees C, which also was observed for MsrB(Tk). Consistent with the low-temperature-specific biochemical properties of MsrAB(Tk), the presence of the protein was greater in T. kodakaraensis cells grown at suboptimal temperatures (60 to 70 degrees C) and could not be detected at 80 to 90 degrees C. We found that the amount of intracellular MsrAB(Tk) protein increased with exposure to higher dissolved oxygen levels, but only at suboptimal growth temperatures. While measuring background rates of the Msr enzyme reactions, we observed significant levels of MetO reduction at high temperatures without enzyme. The occurrence of nonenzymatic MetO reduction at high temperatures may explain the specific absence of Msr homologs in most hyperthermophiles. Together with the fact that the presence of Msr in T. kodakaraensis is exceptional among the hyperthermophiles, the enzyme may represent a novel strategy for this organism to deal with low-temperature environments in which the dissolved oxygen concentrations increase.     

里氏木霉不同糖苷水解酶基因转录水平比较分析

以前期里氏木霉RNA-seq中发现的7个糖苷水解酶基因为对象,分析其不同条件下的表达特性,以期为寻找新的纤维素降解功能酶提供证据。运用生物信息学方法,分析了7个基因可能的编码产物和结构特征。以不同的产纤维素酶菌株（QM 9414、RUT C30）为材料,采用实时荧光定量PCR,对7个糖苷水解酶基因（编号4–10）在各种碳源条件下转录情况与主要的3个纤维素酶基因cbh1,cbh2,egl1（编号1–3）进行了比较分析。信息学分析表明,7个基因编码蛋白分属于GH47（4号、5号）,GH92（6–8号）,GH16（9号）,GH31（10号）糖苷水解酶家族,具有典型的信号肽序列。cbh1,cbh2,egl1基因在纤维素酶诱导条件下,转录水平均表现显著的增加,上调倍数以QM 9414菌株表现的最高。QM 9414菌株中,cbh1,cbh2,egl1基因在纤维素条件下的上调倍数显著高于乳糖,3个基因在RUT C30菌株中的转录水平则显示乳糖条件下上调幅度更大。7个糖苷水解酶基因也存在类似的情况,而且编码α-甘露糖苷酶和内切β-葡聚糖酶的8号、9号基因上调倍数在纤维素酶诱导条件下仅次于纤维素酶基因,而以甘油为碳源条件下,8号、9号基因上调倍数高于纤维素酶基因。4号基因在上述碳源条件下,转录水平变化不大。结果表明：4号基因可能是组成型表达。基因5、6、7、8、9、10的表达呈现明显的菌株和碳源依赖性,且在纤维素酶诱导条件下基本上是和3个纤维素酶基因共转录的。     

Structural and kinetic analysis of an MsrA–MsrB fusion protein from Streptococcus pneumoniae

Methionine sulphoxide reductases (Msr) catalyse the reduction of oxidized methionine to methionine. These enzymes are divided into two classes, MsrA and MsrB, according to substrate specificity. Although most MsrA and MsrB exist as separate enzymes, in some bacteria these two enzymes are fused to form a single polypeptide (MsrAB). Here, we report the first crystal structure of MsrAB from Streptococcus pneumoniae ( Sp MsrAB) at 2.4 Å resolution. Sp MsrAB consists of an N-terminal MsrA domain, a C-terminal MsrB domain and a linker. The linker is composed of 13 residues and contains one 3₁₀-helix and several hydrogen bonds interacting with both MsrA and MsrB domains. Interestingly, our structure includes the MsrB domain complexed with an SHMAEI hexa-peptide that is the N-terminal region of neighbouring MsrA domain. A kinetic analysis showed that the apparent K _m of Sp MsrAB for the R -form-substrate was 20-fold lower than that for the S -form substrate, indicating that the MsrB domain had a much higher affinity for the substrate than the MsrA domain. Our study reveals the first structure of the MsrAB by providing insights into the formation of a disulphide bridge in the MsrB, the structure of the linker region, and the distinct structural nature of active site of each MsrA and MsrB domain.     

Functional analysis of the early chlorosis factor gene

Chlorosis is one of the symptoms of bacterial spot disease caused by Xanthomonas campestris pv. vesicatoria, which induces chlorosis before any other symptoms appear on tomato. We report characterization of a 2.1-kb gene called early chlorosis factor (ecf). The gene ecf encodes a hydrophobic protein with similarity to four other proteins in plant pathogens, including HolPsyAE, and uncharacterized gene products from X. campestris pv. campestris and X. axonopodis pv. citri, and, at the tertiary structure level, to colicin Ia from Escherichia coli. We demonstrate that the associated phenotype is hrp dependent, and that the ecf gene product appears to be translocated to host cells. The gene ecf has no impact on electrolyte leakage or on bacterial growth in planta in response to infection. Concentrated culture filtrates do not produce chlorosis. Study of its role in Xanthomonas spp.-tomato interactions will forward our understanding of symptom production by plant pathogens and allows further investigation into the mechanisms of bacterial virulence and production of symptoms.