Structure and redox properties of the haem centre in the C357M mutant of cytochrome P450cam

The effects of site-specific mutation of the axial cysteine (C357M) to a methionine residue in cytochrome P450cam on the enzyme's coordination geometry and redox potential have been investigated. The absorption spectra of the haem centre in the C357M mutant of the enzyme showed close similarity to those of cytochrome c both in the oxidised and reduced forms. A well-defined absorption peak at 695 nm, similar to that seen in the case of cytochrome c and characteristic of methionine ligation to the ferric haem, was observed. The results indicated that the haem of C357M cytochrome P450cam is possibly axially coordinated to a methionine and a histidine, analogously to cytochrome c. The circular dichroism spectra in the visible and the far-UV regions suggested that the tertiary structure of the haem cavity in the C357M mutant cytochrome P450cam was distinctly different from that in the wild-type enzyme or in cytochrome c, although the secondary structure of the mutant remained identical to that of the wild-type cytochrome P450cam. Comparison of the natures of the CD spectra in the 400 nm and 695 nm regions of the C357M mutant of cytochrome P450cam with those of horse cytochrome c suggested (R) chirality at the sulfur atom of the iron-bound methionine residue in the mutant. The redox potential of the haem centre, estimated by redox titration of the C357M mutant, was found to be +260 mV, which is much higher than that in the wild-type enzyme and similar to the redox potential of cytochrome c. This supported the concept that axial ligation of the haem plays the major role in tuning the redox potential of the haem centre in haem proteins.     

Site-directed alteration of Glu197 and Glu66 in a pyruvoyl-dependent histidine decarboxylase

Site-directed mutagenesis has been used to explore the roleof two carboxylates in the active site of histidine decarboxylasefrom Lactobacillus 30a. The most striking observation is thatconversion of Glu197 to either Gln or Asp causes a major decreasein catalytic rate while enhancing substrate binding. This isconsistent with models based on X-ray diffraction results whichsuggest that the acid may protonate a reaction intermediateduring catalysis. The Asp197 protein undergoes a suicide reactionwith substrate, apparently triggered by inappropriate protonationof the intermediate. This leads to decarboxylation-dependenttransamination which converts the pyruvoyl cofactor to an alanine,inactivating the enzyme. Conversion of Glu66 to Gln affectsparameters of kinetic cooperativity. The mutation fixes theHill number at – 1.5, midway between the pH-dependentvalues of the wild-type enzyme.     

Genetic Encoding of a Photocaged Histidine for Light-Control of Protein Activity

The use of light to control protein function is a critical tool in chemical biology. Here we describe the addition of a photocaged histidine to the genetic code. This unnatural amino acid becomes histidine upon exposure to light and allows for the optical control of enzymes that utilize active-site histidine residues. We demonstrate light-induced activation of a blue fluorescent protein and a chloramphenicol transferase. Further, we genetically encoded photocaged histidine in mammalian cells. We then used this approach in live cells for optical control of firefly luciferase and, Renilla luciferase. This tool should have utility in manipulating and controlling a wide range of biological processes.     

Loop结构引入半胱氨酸对木聚糖酶XynASP热稳定性的影响

为了探究Loop结构引入半胱氨酸对GH11家族木聚糖酶热稳定性的影响,以溶糖曲霉（Aspergillus saccharolyticus）JOP 1030-1木聚糖酶XynASP Loop结构为研究对象,通过定点突变技术,将第95位点天冬酰胺（Asn）突变成半胱氨酸（Cys）,获得突变体XynN95C,并在大肠杆菌（Escherichia coli）BL21（DE3）中诱导表达。酶学性质分析结果表明,突变体XynN95C最适温度为50 ℃,酶活性半衰期t1/240 ℃为38 min,相比野生型XynASP分别提高了5 ℃、18 min,最适pH从6.0降至5.0。另外,XynN95C的金属离子耐受性较强,经Fe3+处理1 h,相对酶活性为93.9%,较野生型（65.9%）明显提高。由此得出,在Loop结构引入半胱氨酸可有效提高木聚糖酶的热稳定性,这为GH11家族木聚糖酶的热稳定性改造又提供一新思路。     

Making Enzymes Suitable for Organic Chemistry by Rational Protein Design

This review outlines recent developments in protein engineering of stereo- and regioselective enzymes, which are of prime interest in organic and pharmaceutical chemistry as well as biotechnology. The widespread application of enzymes was hampered for decades due to limited enantio-, diastereo- and regioselectivity, which was the reason why most organic chemists were not interested in biocatalysis. This attitude began to change with the advent of semi-rational directed evolution methods based on focused saturation mutagenesis at sites lining the binding pocket. Screening constitutes the labor-intensive step (bottleneck), which is the reason why various research groups are continuing to develop techniques for the generation of small and smart mutant libraries. Rational enzyme design, traditionally an alternative to directed evolution, provides small collections of mutants which require minimal screening. This approach first focused on thermostabilization, and did not enter the field of stereoselectivity until later. Computational guides such as the Rosetta algorithms, HotSpot Wizard metric, and machine learning (ML) contribute significantly to decision making. The newest advancements show that semi-rational directed evolution such as CAST/ISM and rational enzyme design no longer develop on separate tracks, instead, they have started to merge. Indeed, researchers utilizing the two approaches have learned from each other. Today, the toolbox of organic chemists includes enzymes, primarily because the possibility of controlling stereoselectivity by protein engineering has ensured reliability when facing synthetic challenges. This review was also written with the hope that undergraduate and graduate education will include enzymes more so than in the past.     

Full-Spectrum Targeted Mutagenesis in Plant and Animal Cells

Directed evolution is a powerful approach for protein engineering and functional studies. However, directed evolution outputs from bacterial and yeast systems do not always translate to higher organisms. In situ directed evolution in plant and animal cells has previously been limited by an inability to introduce targeted DNA sequence diversity. New hypermutation tools have emerged that can generate targeted mutations in plant and animal cells, by recruiting mutagenic proteins to defined DNA loci. Progress in this field, such as the development of CRISPR-derived hypermutators, now allows for all DNA nucleotides within user-defined regions to be altered through the recruitment of error-prone DNA polymerases or highly active DNA deaminases. The further engineering of these mutagenesis systems will potentially allow for all transition and transversion substitutions to be generated within user-defined genomic windows. Such targeted full-spectrum mutagenesis tools would provide a powerful platform for evolving antibodies, enzymes, structural proteins and RNAs with specific desired properties in relevant cellular contexts. These tools are expected to benefit many aspects of biological research and, ultimately, clinical applications.     

Antioxidative Activity and Safety of 50% Ethanolic Red Bean Extract (Phaseolus radiatus L. var. Aurea)

ABSTRACT: This study evaluated the antioxidative activities of 50% ethanolic extract from red bean (Phaseolus radiatus L. var. Aurea). The antioxidative activities, including α,α‐diphenyl‐β‐picryl‐hydrazyl (DPPH) radicals scavenging effects, Fe²⁺‐chelating ability, and reducing power, were studied in vitro. The antioxidative activity was found to increase with the concentration of the extract to a certain extent and then level off as the concentration further increased. Compared with commercial antioxidants, the red bean extract showed less scavenging effect on the DPPH radical and less reducing power than α‐Tocopherol and BHT, but better Fe²⁺‐chelating ability. No mutagenic effect toward any tester strains was found in the 50% ethanolic extract of red bean.     

香蕉酒嗜杀野生酿酒酵母的筛选

该研究采用从香蕉酒醪中初筛获得的嗜杀野生酿酒酵母进行热冲击处理和紫外诱变(灯管功率20W,波长254nm,照射距离为50cm);经2,3,5-氯化三苯基四氮唑(TTC)的显色筛选,获得了5株呈深红色菌落的突变株;通过麦芽汁发酵对比,选定一株优良菌株.该菌株在麦芽汁糖度为10°BX,温度为20℃的条件下发酵7d,发酵液最终的糖度为1.71g/L,最终pH值为3.8,可溶性固形物为2.86％,酒精体积分数为4.2％vol.