Giuseppe Perugino

A novel thermophilic Glycosynthase that effects branching glycosylation

A novel thermophilic glycosynthase that effects branching glycosylation has been obtained by mutation of the nucleophile in the active site of the glycosidase from Sulfolobus solfataricus. Two methods for the use of this mutant are reported.

Reverse gyrase and genome stability in hyperthermophilic organisms.

Reverse gyrase is a DNA topoisomerase that is peculiar in many aspects: it has the unique ability to introduce positive supercoils into DNA molecules; it comprises a type IA topoisomerase fused to a helicase-like domain; although it is a type IA topoisomerase, its reaction is ATP-dependent; and it is the only hyperthermophile-specific protein. All these features have made reverse gyrase the subject of biochemical, structural and functional studies, although they have not shed complete light on the evolution, mechanism and function of this distinctive enzyme. In the present article, we review the latest progress on structure-function relationships of reverse gyrase, and discuss old and recent data linking reverse gyrase to DNA stability, protection and repair in hyperthermophilic organisms.

Dissection of reverse gyrase activities: insight into the evolution of a thermostable molecular machine

Reverse gyrase is a peculiar DNA topoisomerase, specific of thermophilic microorganisms, which induces positive supercoiling into DNA molecules in an ATP-dependent reaction. It is a modular enzyme and comprises an N-terminal helicase-like module fused to a C-terminal topoisomerase IA-like domain. The exact molecular mechanism of this unique reaction is not understood, and a fundamental mechanistic question is how its distinct steps are coordinated. We studied the cross-talk between the components of this molecular motor and probed communication between the DNA-binding sites and the different activities (DNA relaxation, ATP hydrolysis and positive supercoiling). We show that the isolated ATPase and topoisomerase domains of reverse gyrase form specific physical interactions, retain their own DNA binding and enzymatic activities, and when combined cooperate to achieve the unique ATP-dependent positive supercoiling activity. Our results indicate a mutual effect of both domains on all individual steps of the reaction. The C-terminal domain shows ATP-independent topoisomerase activity, which is repressed by the N-terminal domain in the full-length enzyme; experiments with the isolated domains showed that the C-terminal domain has stimulatory influence on the ATPase activity of the N-terminal domain. In addition, the two domains showed a striking reciprocal thermostabilization effect.

Enzymatic synthesis of oligosaccharides by two glycosyl hydrolases of Sulfolobus solfataricus

Abstract. The importance of carbohydrates in a variety of biological functions is the reason that interest has recently increased in these compounds as possible components of therapeutic agents. Thus, the need for a technique allowing the easy synthesis of carbohydrates and glucoconjugates is an emerging challenge for chemists and biologists involved in this field. At present, enzymatic synthesis has resulted in the most promising approach for the production of complex oligosaccharides. In this respect, the enzymological characteristics of the catalysts, in term of regioselectivity, substrate specificity, and operational stability, are of fundamental importance to improve the yields of the process and to widen the repertoire of the available products. Here, two methods of oligosaccharide synthesis performed by a glycosynthase and by an α-xylosidase from the hyperthermophilic archaeon Sulfolobus solfataricus are briefly reviewed. The approaches used and the biodiversity of the catalysts together are key features for their possible utilization in the synthesis of oligosaccharides.

Activity and Regulation of Archaeal DNA Alkyltransferase CONSERVED PROTEIN INVOLVED IN REPAIR OF DNA ALKYLATION DAMAGE

Background: DNA alkyltransferases repair mutagenic and carcinogenic alkylation DNA lesions. Results: A thermophilic archaeal DNA alkyltransferase is degraded after alkylation in vivo. A novel assay is applied to study its activity in vitro. Conclusion: The archaeal DNA alkyltransferase shows structure, activity, and in vivo regulation similar to its human homolog. Significance: The function and regulation of DNA alkyltransferases might be conserved from archaea to humans. Agents that form methylation adducts in DNA are highly mutagenic and carcinogenic, and organisms have evolved specialized cellular pathways devoted to their repair, including DNA alkyltransferases. These are proteins conserved in eucarya, bacteria and archaea, acting by a unique reaction mechanism, which leads to direct repair of DNA alkylation damage and irreversible protein alkylation. The alkylated form of DNA alkyltransferases is inactive, and in eukaryotes, it is rapidly directed to degradation. We report here in vitro and in vivo studies on the DNA alkyltransferase from the thermophilic archaeon Sulfolobus solfataricus (SsOGT). The development of a novel, simple, and sensitive fluorescence-based assay allowed a careful characterization of the SsOGT biochemical and DNA binding activities. In addition, transcriptional and post-translational regulation of SsOGT by DNA damage was studied. We show that although the gene transcription is induced by alkylating agent treatment, the protein is degraded in vivo by an alkylation-dependent mechanism. These experiments suggest a striking conservation, from archaea to humans, of this important pathway safeguarding genome stability.

A comparative infrared spectroscopic study of glycoside hydrolases from extremophilic archaea revealed different molecular mechanisms of adaptation to high temperatures

The identification of the determinants of protein thermal stabilization is often pursued by comparing enzymes from hyperthermophiles with their mesophilic counterparts while direct structural comparisons among proteins and enzymes from hyperthermophiles are rather uncommon. Here, oligomeric β‐glycosidases from the hyperthermophilic archaea Sulfolobus solfataricus (Ssβ‐gly), Thermosphaera aggregans (Taβ‐gly), and Pyrococcus furiosus (Pfβ‐gly), have been compared. Studies of FTIR spectroscopy and kinetics of thermal inactivation showed that the three enzymes had similar secondary structure composition, but Ssβ‐gly and Taβ‐gly (temperatures of melting 98.1 and 98.4°C, respectively) were less stable than Pfβ‐gly, which maintained its secondary structure even at 99.5°C. The thermal denaturation of Pfβ‐gly, followed in the presence of SDS, suggested that this enzyme is stabilized by hydrophobic interactions. A detailed inspection of the 3D‐structures of these enzymes supported the experimental results: Ssβ‐gly and Taβ‐gly are stabilized by a combination of ion‐pairs networks and intrasubunit S‐S bridges while the increased stability of Pfβ‐gly resides in a more compact protein core. The different strategies of protein stabilization give experimental support to recent theories on thermophilic adaptation and suggest that different stabilization strategies could have been adopted among archaea. Proteins 2007.

Highly Productive Autocondensation and Transglycosylation Reactions with Sulfolobus solfataricus Glycosynthase

Transglycosylation reactions (autocondensation of the substrate or transfer of the glycon donor moiety to different acceptors) with the hyperthermophilic glycosynthase from Sulfolobus solfataricus acting in dilute sodium formate buffer at pH 4.0 are reported; the use of 4‐nitrophenyl β‐glucopyranoside as both donor and acceptor in the self‐transfer reaction and a highly productive reaction with 1.1u2009M 2‐nitrophenyl β‐glucopyranoside were possible. Interesting effects, governed by the anomeric configuration and lipophilicity of heteroacceptors, on the regioselectivity and yield of reactions were found for the first time with this enzyme and are discussed. The results demonstrate the unexplored synthetic potential of this glycosynthase; the tuning of the reaction conditions and the choice of different donors/acceptors can lead to products of applicative interest.

Biochemical and Structural Studies of the Mycobacterium tuberculosis O6-Methylguanine Methyltransferase and Mutated Variants

Mycobacterium tuberculosis displays remarkable genetic stability despite continuous exposure to the hostile environment represented by the hosts infected macrophages. Similarly to other organisms, M. tuberculosis possesses multiple systems to counteract the harmful potential of DNA alkylation. In particular, the suicidal enzyme O(6)-methylguanine-DNA methyltransferase (OGT) is responsible for the direct repair of O(6)-alkylguanine in double-stranded DNA and is therefore supposed to play a central role in protecting the mycobacterial genome from the risk of G · C-to-A · T transition mutations. Notably, a number of geographically widely distributed M. tuberculosis strains shows nonsynonymous single-nucleotide polymorphisms in their OGT-encoding gene, leading to amino acid substitutions at position 15 (T15S) or position 37 (R37L) of the N-terminal domain of the corresponding protein. However, the role of these mutations in M. tuberculosis pathogenesis is unknown. We describe here the in vitro characterization of M. tuberculosis OGT (MtOGT) and of two point-mutated versions of the protein mimicking the naturally occurring ones, revealing that both mutated proteins are impaired in their activity as a consequence of their lower affinity for alkylated DNA than the wild-type protein. The analysis of the crystal structures of MtOGT and MtOGT-R37L confirms the high level of structural conservation of members of this protein family and provides clues to an understanding of the molecular bases for the reduced affinity for the natural substrate displayed by mutated MtOGT. Our in vitro results could contribute to validate the inferred participation of mutated OGTs in M. tuberculosis phylogeny and biology.

Inhibition of translesion DNA polymerase by archaeal reverse gyrase

Reverse gyrase is a unique DNA topoisomerase endowed with ATP-dependent positive supercoiling activity. It is typical of microorganisms living at high temperature and might play a role in maintenance of genome stability and repair. We have identified the translesion DNA polymerase SsoPolY/Dpo4 as one partner of reverse gyrase in the hyperthermophilic archaeon Sulfolobus solfataricus. We show here that in cell extracts, PolY and reverse gyrase co-immunoprecipitate with each other and with the single strand binding protein, SSB. The interaction is confirmed in vitro by far-western and Surface Plasmon Resonance. In functional assays, reverse gyrase inhibits PolY, but not the S. solfataricus B-family DNA polymerase PolB1. Mutational analysis shows that inhibition of PolY activity depends on both ATPase and topoisomerase activities of reverse gyrase, suggesting that the intact positive supercoiling activity is required for PolY inhibition. In vivo, reverse gyrase and PolY are degraded after induction of DNA damage. Inhibition by reverse gyrase and degradation might act as a double mechanism to control PolY and prevent its potentially mutagenic activity when undesired. Inhibition of a translesion polymerase by topoisomerase-induced modification of DNA structure may represent a previously unconsidered mechanism of regulation of these two-faced enzymes.

Positive supercoiling in thermophiles and mesophiles: of the good and evil

DNA supercoiling plays essential role in maintaining proper chromosome structure, as well as the equilibrium between genome dynamics and stability under specific physicochemical and physiological conditions. In mesophilic organisms, DNA is negatively supercoiled and, until recently, positive supercoiling was considered a peculiar mark of (hyper)thermophilic archaea needed to survive high temperatures. However, several lines of evidence suggest that negative and positive supercoiling might coexist in both (hyper)thermophilic and mesophilic organisms, raising the possibility that positive supercoiling might serve as a regulator of various cellular events, such as chromosome condensation, gene expression, mitosis, sister chromatid cohesion, centromere identity and telomere homoeostasis.