Biochemical Journal - BJ Structure

New insights into the structure of the reaction centre from Blastochloris viridis: evolution in the laboratory

2012-02-15

Newly determined crystal structures of the photosynthetic RC (reaction centre) from two substrains of the non-sulfur purple bacterium Blastochloris viridis strain DSM 133, together with analysis of their gene sequences, has revealed intraspecies evolutionary changes over a period of 14 years. Over 100 point mutations were identified between these two substrains in the four genes encoding the protein subunits of the RC, of which approximately one-fifth resulted in a total of 16 amino acid changes. The most interesting difference was in the M subunit where the change from a leucine residue to glycine in the carotenoid-binding pocket allowed NS5 (1,2-dihydroneurosporene) to adopt a more sterically favoured conformation, similar to the carotenoid conformation found in other related RCs. The results of the present study, together with a high rate of mutations in laboratory bacterial cultures described recently, suggest that bacteria evolve faster than has been generally recognized. The possibility that amino acid changes occur within protein sequences, without exhibiting any immediately observable phenotype, should be taken into account in studies that involve long-term continuous growth of pure bacterial cultures. The Blc. viridis RC is often studied with sophisticated biophysical techniques and changes such as those described here may well affect their outcome. In other words, there is a danger that laboratory-to-laboratory variation could well be due to different groups not realising that they are actually working with slightly different proteins. A way around this problem is suggested.

The pH sensor of the plant K+-uptake channel KAT1 is built from a sensory cloud rather than from single key amino acids

2012-02-15

The uptake of potassium ions (K⁺) accompanied by an acidification of the apoplasm is a prerequisite for stomatal opening. The acidification (approximately 2–2.5 pH units) is perceived by voltage-gated inward potassium channels (K_in) that then can open their pores with lower energy cost. The sensory units for extracellular pH in stomatal K_in channels are proposed to be histidines exposed to the apoplasm. However, in the Arabidopsis thaliana stomatal K_in channel KAT1, mutations in the unique histidine exposed to the solvent (His²⁶⁷) do not affect the pH dependency. We demonstrate in the present study that His²⁶⁷ of the KAT1 channel cannot sense pH changes since the neighbouring residue Phe²⁶⁶ shifts its pK_a to undetectable values through a cation–π interaction. Instead, we show that Glu²⁴⁰ placed in the extracellular loop between transmembrane segments S5 and S6 is involved in the extracellular acid activation mechanism. Based on structural models we propose that this region may serve as a molecular link between the pH- and the voltage-sensor. Like Glu²⁴⁰, several other titratable residues could contribute to the pH-sensor of KAT1, interact with each other and even connect such residues far away from the voltage-sensor with the gating machinery of the channel.

Staphylococcus aureus DinG, a helicase that has evolved into a nuclease

2012-02-15

DinG (damage inducible gene G) is a bacterial superfamily 2 helicase with 5′→3′ polarity. DinG is related to the XPD (xeroderma pigmentosum complementation group D) helicase family, and they have in common an FeS (iron–sulfur)-binding domain that is essential for the helicase activity. In the bacilli and clostridia, the DinG helicase has become fused with an N-terminal domain that is predicted to be an exonuclease. In the present paper we show that the DinG protein from Staphylococcus aureus lacks an FeS domain and is not a DNA helicase, although it retains DNA-dependent ATP hydrolysis activity. Instead, the enzyme is an active 3′→5′ exonuclease acting on single-stranded DNA and RNA substrates. The nuclease activity can be modulated by mutation of the ATP-binding cleft of the helicase domain, and is inhibited by ATP or ADP, suggesting a modified role for the inactive helicase domain in the control of the nuclease activity. By degrading rather than displacing RNA or DNA strands, the S. aureus DinG nuclease may accomplish the same function as the canonical DinG helicase.

A preliminary characterization of the cytosolic glutathione transferase proteome from Drosophila melanogaster

Chonticha Saisawang, Jantana Wongsantichon and Albert J. Ketterman — 2012-02-15

The cytosolic GST (glutathione transferase) superfamily has been annotated in the Drosophila melanogaster genome database. Of 36 genes, four undergo alternative splicing to yield a total of 41 GST proteins. In the present study, we have obtained the 41 transcripts encoding proteins by RT (reverse transcription)–PCR using RNA template from Drosophila S2 cells, an embryonic cell line. This observation suggests that all of the annotated DmGSTs (D. melanogaster GSTs) in the proteome are expressed in the late embryonic stages of D. melanogaster. To avoid confusion in naming these numerous DmGSTs, we have designated them following the universal GST nomenclature as well as previous designations that fit within this classification. Furthermore, in the cell line, we identified an apparent processed pseudogene, gste8, in addition to two isoforms from the Delta class that have been published previously. Only approximately one-third of the expressed DmGSTs could be purified by conventional GSH affinity chromatography. The diverse kinetic properties as well as physiological substrate specificity of the DmGSTs are such that each individual enzyme displayed a unique character even compared with members from the same class.