PREFACE

This volume is dedicated to the memory of the Nobel Laureate Chris-
tian B. Anfinsen. In typical fashion he had the foresight to determine
that a book dealing just with proteins from hyperthermophiles was time-
ly, even though some of us thought that such a book might be premature.
The following pages represent the product of Chris’s original idea. The
reader will no doubt conclude that he was right. Moreover, without
Chris's urging, encouragement, and relentless enthusiasm at the initia-
tion of this project, I doubt if a Preface would be needed at this time.
Suffice it to say that this volume represents a very small monument to
a man who achieved in one lifetime what few dream of in many. ,

A volume dedicated to the properties of enzymes and proteins derived
from the so-called hyperthermophiles would not have been possible
even five years ago. Hyperthermophiles are defined as microorganisms
that can grow at temperatures of 90°C and above. They were discovered
in shallow marine volcanic vents in the early 1980s by Karl Stetter and
co-workers. Mainly through their pioneering studies, the field has greatly
expanded over the past decade, and more than thirty species of hyper-
thermophile are known at present. However, as might be expected,
because of the problems with cultivating these organisms, it was some
time after they were discovered that the first proteins were purified
from them. Indeed, the initial examples of hyperthermophiles were
autotrophic, and their growth was obligately dependent on reducing
elemental sulfur to hydrogen sulfide. Typically, they did not reach high
cell densities. It was not until the mid-1980s that heterotrophs able to
grow well in the absence of sulfur were isolated, such as species of
Pyrococcus and Thermotoga. With these organisms, large-scale cultures
became feasible, and sufficient biomass could be routinely obtained for
protein purifications. Such species have been the source of most of the
hyperthermophilic proteins characterized so far.

With the availability of proteins from hyperthermophiles, researchers
in this field could obviously begin to address the fundamental question
of how they are stabilized at temperatures near and even above the
normal boiling point. With the development of this field during the
1980s, however, it became clear that the classification of the organisms
themselves raised other interesting issues. Specifically, the seminal re-
search of Carl Woese and co-workers in the late 1970s led to the descrip-

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xii PREFACE

tion, on the basis of 16S rRNA analyses, of a new “‘domain’’ of life—the
archaea, formerly archaebacteria. Moreover, subsequent work by Woese
indicated that the archaea domain had an ancestor in common with
eukaryotes which was not shared by bacteria. Consequently, as each new
hyperthermophilic species was isolated, virtually all were found to be
members of the archaea. In fact, at present, only two genera of hyperther-
mophiles are known, Thermotoga and Aquifex, that are classified as bacte-
ria. The ecology and taxonomy of the hyperthermophilic archaea are
discussed in the first chapter by John Baross and James Holden. These
researchers also present the current status of a somewhat unique topic—
hyperthermophilic heat-shock proteins. Remarkably, these organisms
do show both a “‘hot’”’ and ‘“‘cold”’ stress response, and the nature of
the proteins involved and how they function are just beginning to be ex-
plored.

The fact that most hyperthermophilic organisms are archaea rather
than bacteria raises several interesting questions regarding the nature
of their biochemical pathways and the enzymes and proteins involved.
Do these organisms utilize bacterial-type metabolic routes for utilizing
growth substrates and for conserving energy, or are they more eukaryotic-
like? The subsequent two chapters by Robert Maier and by Arnulf Kletzin
and me discuss these issues with reference to the respiratory chains
of sulfur-respiring organisms and to the fermentative pathways of the
hyperthermophilic archaea, respectively. We also discuss the first struc-
tural information to be obtained on both a hyperthermophilic protein
(rubredoxin) and a hyperthermophilic enzyme (aldehyde ferredoxin
oxidoreductase). Rainer Jaenicke and co-workers provide a contrasting
description of what is known about the enzymology of carbohydrate
fermentation in the hyperthermophilic bacterium, Thermotoga maritima.
The ability to metabolize complex carbohydrates and proteinaceous
materials is virtually a characteristic of the hyperthermophiles, including
the majority of the archaea and T. maritima. The enzymes involved
have significant biotechnological potential, and the properties of those
purified so far are discussed by Robert Kelly and co-workers. This is
followed by a summary by Jocelyne DiRuggiero and Frank T. Robb of
what is known about the enzymes involved in the primary metabolism
of nitrogen-containing compounds.

An increasing number of hyperthermophilic organisms are being iso-
lated from near deep sea hydrothermal vents. These occur several kilo-
meters below sea level, and organisms living in their vicinity are exposed
to two extreme conditions—high hydrostatic pressure (approximately
100 atm per 1 km depth) as well as high temperature. Although no
obligately barophilic hyperthermophile has been isolated so far, there

PREFACE xiii

is evidence that at least some hyperthermophilic enzymes are stabilized
and/or activated by high pressure. Douglas Clark and co-workers discuss
the recent data on this subject. Of course, the main ‘‘claim to fame”
of hyperthermophiles in a practical sense is that they have provided
heat-stable polymerases for the PCR reaction. Although the prototypical
enzyme, Taq polymerase, was obtained from what is now considered
to be a moderately thermophilic organism, the Tag enzyme has been
superseded to some extent by a variety of polymerases from the hyperth-
ermophiles. In addition to their commercial significance, these enzymes
have also given new insights into protein structure and function, particu-
larly with the phenomena of inteins and exteins. The current diversity
of DNA polymerases is described by Francine Perler and colleagues. Of
course, any analysis of such enzymes assumes that their substrate, DNA,
is stable at the growth temperature of the organisms from which they
were obtained. Hence, John Reeve and co-workers discuss the problems
that hyperthermophiles have in maintaining DNA in a double helical
form near and above 100°C and the likely role of histone-type proteins.

Key aspects of all the research discussed in this volume are the mecha-
nisms of protein stability and the evolutionary significance of hyperther-
mophilic enzymes and proteins. As will become apparent, meaningful
insights into the stability issue have yet to be elucidated. Although hyper-
thermophilic proteins are for the most part exceedingly thermostable
in vitro, this property is apparently conferred by the same intraprotein
interactions that stabilize mesophilic proteins. The problem is that the
differences between the hyperthermophilic and mesophilic versions are
not dramatic, or even obvious, and the ‘“‘holy grail’’ of elucidating a
general mechanism by which a given mesophilic protein can be con-
yerted into a hyperthermophilic one is not likely to be found in the
near future. In contrast, the phylogenetic aspects of hyperthermophilic
proteins are providing new insights into likely pathways of enzyme evolu-
tion, although the results are not always congruent with phylogenetic
relationships determined by nucleic acid analyses. Future studies of
proteins from both known hyperthermophiles and those yet to be discov-
ered will no doubt provide deeper insights into both the mechanisms
of protein stability and enzyme evolution.

MICHAEL W. W. ADAMS