J. Molec. Microbiol. Biotechnol. (1999) 1(1): 101-105. Protein Stability at High PressureJMMB and SymposiumTemperature 101 Adaptation of Proteins from Hyperthermophiles to High Pressure and High Temperature Frank T. Robb1*, and Douglas S. Clark2 application of novel methods of analysis. For example, two key enzymes in glycolysis, phosphofructokinase and 1Center of Marine Biotechnology, University of Maryland hexokinase, remained undiscovered in extracts of the Biotechnology Institute, Baltimore, MD 21202, USA hyperthermophile Pyrococcus furiosus because it was 2Department of Chemical Engineering, University of anticipated that they would require ATP. It transpires that California, Berkeley, USA enzymes are active and highly expressed, but require ADP (Kengen et al., 1995), presumably due to the higher stability of ADP compared with ATP. This review focuses on Abstract mechanisms of stabilization of enzymes with novel catalytic specificities, unusual thermal stability, and the ability to Further clarification of the adaptations permitting the withstand, and in many cases to gain enhanced persistence of life at temperatures above 100 °C thermostability under elevated hydrostatic pressure. The depends in part on the analysis of adaptive latter is an unusual and intriguing feature of many proteins mechanisms at the protein level. The hypertherm- from thermophiles and recent work suggests that the ophiles include both Bacteria and Archaea, although structural stability of proteins in the 100-130 °C range is the majority of isolates growing at or above 100 °C are achieved by quite generic adaptive mechanisms. We will Archaea. Newly described adaptive features of review the current field of protein stability at very high hyperthermophiles include proteins whose structural temperatures and discuss recent findings on the theoretical integrity persists at temperatures up to 200 °C, and basis for pressure stabilization of proteins. under elevated hydrostatic pressure, which in some cases adds significant increments of stability. Mechanisms of Hyperstability Introduction The known strategies for maintaining stability at high temperatures are summarized in Table 1. Five classes of It is now very well established that microbial growth can adaptations are discussed below. occur at temperatures well above 100 °C. The recently described microorganism with the highest recorded growth 1. Amino Acid Composition temperature of 113.5 °C, is the chemolithoautotrophic Although many formulae for compositional bias in archaeon Pyrolobus fumarii (Blochl et al., 1997). hyperstable proteins have been suggested, there appear Hyperthermophiles, which are defined as organisms with to be few answers to the quest for a thermoadapted amino maximal growth temperatures above 90 °C (Adams, 1994), acid composition. On the contrary, hyperstable proteins, are widely distributed in hydrothermal environments in like their counterparts in mesophiles, are composed of the terrestrial as well marine and abyssal vent systems. The same 20 amino acid set found in mesophiles. These amino group includes 21 archaeal genera, and two bacterial acids undergo accelerated covalent modification at the high genera with diverse growth physiology including both temperatures, pressures and extremes of pH that many heterotrophs and chemoautotrophs (Stetter, 1996; Robb thermophiles and hyperthermophiles can withstand (Hensel et al., 1995). The archaeal hyperthermophiles have et al., 1992). At temperatures beyond 100 °C, the stability exceptionally high growth temperature limits, unique, of common amino acids declines in the following order: molecular characteristics, such as ether-linked lipids and (Val,Leu)>Ile>Tyr>Lys>His>Met>Thr>Ser> Trp>(Asp, Glu, eukaryotic transcription and translation factors (Robb et Arg, Cys) (Jaenicke 1991; Jaenicke 1996a; Jaenicke and al. 1995), and unusual enzyme chemistry, such as the Bohm, 1998). Since many of the hyperthermophiles are presence of multiple tungsten-containing enzymes (Kletzin relatively slow growing, the half lives of some of the more and Adams, 1996). The unusual adaptive responses of the labile amino acids such as Cys, Arg, Gln, Asn are hyperthermophiles include heat shock proteins of the hsp60 considerably shorter than the generation times of the family (Trent, 1996; Trent et al., 1997; Kagawa et al., 1995) organisms (Bernhardt et al., 1984). Common types of which are inducible by supraoptimal temperatures, and chemical alterations include deamidation, ß-oxidation, reverse gyrase which installs positive supercoils in DNA Maillard reactions, hydrolysis, and disulfide interchange, (Guipaud et al., 1997; Borges et al., 1997). etc (Jaenicke and Bohm, 1998). Many of the amino acids The ongoing release of genomic sequence data from are more stable upon internalization in the hydrophobic hyperthermophiles will continue to accelerate the discovery core of the proteins than in free solution (Hensel et al., of thermostable proteins, however understanding the 1992), however the frequencies of occurrence of Cys, Asn, functional adaptations of these proteins requires the Gln and Asp is significantly lowered in the hyperstable variants of some common proteins. The genomic sequences that are determined or in progress will continue *For correspondence. Email [email protected]; Tel. 410-234 8870; Fax. 410-234 8896. to make available a flood of deduced protein sequences, © 1999 Horizon Scientific Press Further Reading Caister Academic Press is a leading academic publisher of advanced texts in microbiology, molecular biology and medical research. Full details of all our publications at caister.com • MALDI-TOF Mass Spectrometry in Microbiology Edited by: M Kostrzewa, S Schubert (2016) www.caister.com/malditof • Aspergillus and Penicillium in the Post-genomic Era Edited by: RP Vries, IB Gelber, MR Andersen (2016) www.caister.com/aspergillus2 • The Bacteriocins: Current Knowledge and Future Prospects Edited by: RL Dorit, SM Roy, MA Riley (2016) www.caister.com/bacteriocins • Omics in Plant Disease Resistance Edited by: V Bhadauria (2016) www.caister.com/opdr • Acidophiles: Life in Extremely Acidic Environments Edited by: R Quatrini, DB Johnson (2016) www.caister.com/acidophiles • Climate Change and Microbial Ecology: Current Research and Future Trends Edited by: J Marxsen (2016) www.caister.com/climate • Biofilms in Bioremediation: Current Research and Emerging Technologies • Flow Cytometry in Microbiology: Technology and Applications Edited by: G Lear (2016) www.caister.com/biorem Edited by: MG Wilkinson (2015) www.caister.com/flow • Microalgae: Current Research and Applications • Probiotics and Prebiotics: Current Research and Future Trends Edited by: MN Tsaloglou (2016) www.caister.com/microalgae Edited by: K Venema, AP Carmo (2015) www.caister.com/probiotics • Gas Plasma Sterilization in Microbiology: Theory, Applications, Pitfalls and New Perspectives • Epigenetics: Current Research and Emerging Trends Edited by: H Shintani, A Sakudo (2016) Edited by: BP Chadwick (2015) www.caister.com/gasplasma www.caister.com/epigenetics2015 • Virus Evolution: Current Research and Future Directions • Corynebacterium glutamicum: From Systems Biology to Biotechnological Applications Edited by: SC Weaver, M Denison, M Roossinck, et al. (2016) www.caister.com/virusevol Edited by: A Burkovski (2015) www.caister.com/cory2 • Arboviruses: Molecular Biology, Evolution and Control Edited by: N Vasilakis, DJ Gubler (2016) • Advanced Vaccine Research Methods for the Decade of www.caister.com/arbo Vaccines Edited by: F Bagnoli, R Rappuoli (2015) • Shigella: Molecular and Cellular Biology www.caister.com/vaccines Edited by: WD Picking, WL Picking (2016) www.caister.com/shigella • Antifungals: From Genomics to Resistance and the Development of Novel Agents • Aquatic Biofilms: Ecology, Water Quality and Wastewater Edited by: AT Coste, P Vandeputte (2015) Treatment www.caister.com/antifungals Edited by: AM Romaní, H Guasch, MD Balaguer (2016) www.caister.com/aquaticbiofilms • Bacteria-Plant Interactions: Advanced Research and Future Trends Edited by: J Murillo, BA Vinatzer, RW Jackson, et al. (2015) • Alphaviruses: Current Biology www.caister.com/bacteria-plant Edited by: S Mahalingam, L Herrero, B Herring (2016) www.caister.com/alpha • Aeromonas Edited by: J Graf (2015) • Thermophilic Microorganisms www.caister.com/aeromonas Edited by: F Li (2015) www.caister.com/thermophile • Antibiotics: Current Innovations and Future Trends Edited by: S Sánchez, AL Demain (2015) www.caister.com/antibiotics • Leishmania: Current Biology and Control Edited by: S Adak, R Datta (2015) www.caister.com/leish2 • Acanthamoeba: Biology and Pathogenesis (2nd edition) Author: NA Khan (2015) www.caister.com/acanthamoeba2 • Microarrays: Current Technology, Innovations and Applications Edited by: Z He (2014) www.caister.com/microarrays2 • Metagenomics of the Microbial Nitrogen Cycle: Theory, Methods and Applications Edited by: D Marco (2014) www.caister.com/n2 Order from caister.com/order 102 Robb and Clark Table 1. Synopsis of Mechanisms of Extreme Thermostability in Proteins from Hyperthermophiles Organism (Topt) Thermostability feature Protein Reference Pyrococcus furiosus (100 °C) Mg++ requirement KCl (1M) Acetyl-CoA synthetase (ADP-forming) Glasemacher et al., 1997 P. furiosus (100 °C) Salt bridges Glutamate dehydrogenase Klump et al., 1992; Vetriani et al., 1998 Sulfolobus acidocaldarius (85 °C) Lysine monomethylation Sac7d McCrary et al., 1996 NOT salt bridges S. acidocaldarius (85 °C) The data conforms