A growing body of data suggests that protein motion plays an important role in enzyme catalysis. Two highly conserved hydrophobic active site residues in the cofactor-binding pocket of ht-ADH (Leu176 and V260) have been mutated to a series of hydrophobic side chains of smaller size, as well as one deletion mutant, L176Δ. Mutations decrease kcat and increase KM(NAD+). Most of the observed decreases in effects on kcat at pH 7.0 are due to an upward shift in the optimal pH for catalysis; a simple electrostatic model is invoked that relates the change in pKa to the distance between the positively charged nicotinamide ring and bound substrate. Structural modeling of the L176Δ and V260A variants indicates the development of a cavity behind the nicotinamide ring without any significant perturbation of the secondary structure of the enzyme relative to the wild-type. Primary kinetic isotope effects (KIEs) are modestly increased for all mutants. Above the dynamical transition at 30 °C for ht-ADH (Kohen et al., Nature (1999) 399, 496), the temperature dependence of the KIE is seen to increase with decreasing side chain volume at positions 176 and 260. Additionally, the relative trends in the temperature dependence of the KIE above and below 30 °C appear reversed for the cofactor-binding pocket mutants in relation to wild-type protein. The aggregate results are interpreted in the context of a full tunneling model of enzymatic hydride transfer that incorporates both protein conformational sampling (preorganization) and active site optimization of tunneling (reorganization). The reduced temperature dependence of the KIE in the mutants below 30 °C indicates that at low temperature, the enzyme adopts conformations refractory to donor-acceptor distance sampling.
Two
single-tryptophan variants were generated in a thermophilic
alcohol dehydrogenase with the goal of correlating temperature-dependent
changes in local fluorescence with the previously demonstrated catalytic
break at ca. 30 °C (Kohen et al., Nature1999, 399, 496). One tryptophan variant,
W87in, resides at the active site within van der Waals contact of
bound alcohol substrate; the other variant, W167in, is a remote-site
surface reporter located >25 Å from the active site. Picosecond-resolved
fluorescence measurements were used to analyze fluorescence lifetimes,
time-dependent Stokes shifts, and the extent of collisional quenching
at Trp87 and Trp167 as a function of temperature. A subnanosecond
fluorescence decay rate constant has been detected for W87in that
is ascribed to the proximity of the active site Zn2+ and
shows a break in behavior at 30 °C. For the remainder of the
reported lifetime measurements, there is no detectable break between
10 and 50 °C, in contrast with previously reported hydrogen/deuterium
exchange experiments that revealed a temperature-dependent break analogous
to catalysis (Liang et al., Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 9556). We conclude that
the motions that lead to the rigidification of ht-ADH below 30 °C
are likely to be dominated by global processes slower than the picosecond
to nanosecond motions measured herein. In the case of collisional
quenching of fluorescence by acrylamide, W87in and W167in behave in
a similar manner that resembles free tryptophan in water. Stokes shift
measurements, by contrast, show distinctive behaviors in which the
active-site tryptophan relaxation is highly temperature-dependent,
whereas the solvent-exposed tryptophan’s dynamics are temperature-independent.
These data are concluded to reflect a significantly constrained environment
surrounding the active site Trp87 that both increases the magnitude
of the Stokes shift and its temperature-dependence. The results are
discussed in the context of spatially distinct differences in enthalpic
barriers for protein conformational sampling that may be related to
catalysis.
Time-resolved fluorescence dynamics
are investigated in two mutants
of a thermophilic alcohol dehydrogenase (ht-ADH): Y25A (at the dimer
interface) and V260A (at the cofactor-binding domain). These residues,
ca. 32 Å apart, are shown to exhibit opposing low-temperature
effects on the hydride tunneling step. Using single-tryptophan constructs
at the active site (Trp87) and a remote, surface-exposed site (Trp167),
time-dependent Stokes shifts and collisional quenching data allow
an analysis of intra-protein dynamical communication. A double mutant,
Y25A:V260A, was also inserted into each single-Trp construct
and analyzed accordingly. None of the mutations affect fluorescence
lifetimes, Stokes shift relaxation rates, and quenching data for the
surface-exposed Trp167 to an appreciable extent. By contrast, fluorescent
probes of the active-site tryptophan 87 reveal distinctive forms of
dynamical communication. Stokes shifts show that the distal Y25A increases
active-site flexibility, V260A introduces a temperature-dependent
equilibration process not previously reported by such measurements,
and the double mutant (Y25A:V260A) eliminates the temperature-dependent
transition sensed by the active-site tryptophan in the presence of
V260A. Collisional quenching data at Trp87 further show a structural
change in the active-site environment/solvation for V260A. In the
aggregate, the temperature dependencies of the fluorescence data are
distinct from the breaks in behavior previously reported for catalysis
and hydrogen/deuterium exchange, attributed to time scales for the
interconversion of protein conformational substates that are slower
and more global than the local motions monitored within. An extended
network of dynamical communication between the protein dimer surface
and substrate- and cofactor-binding domains emerges from the flourescent
data.
Protein
dynamics on the microsecond (μs) time scale were
investigated by temperature-jump fluorescence spectroscopy as a function
of temperature in two variants of a thermophilic alcohol dehydrogenase:
W87F and W87F:H43A. Both mutants exhibit a fast, temperature-independent
μs decrease in fluorescence followed by a slower full recovery
of the initial fluorescence. The results, which rule out an ionizing
histidine as the origin of the fluorescence quenching, are discussed
in the context of a Trp49-containing dimer interface that acts as
a conduit for thermally activated structural change within the protein
interior.
BackgroundGeranylgeranyl reductase (GGR) is a flavin-containing redox enzyme that hydrogenates a variety of unactivated polyprenyl substrates, which are further processed mostly for lipid biosynthesis in archaea or chlorophyll biosynthesis in plants. To date, only a few GGR genes have been confirmed to reduce polyprenyl substrates in vitro or in vivo.ResultsIn this work, we aimed to expand the confirmed GGR activity space by searching for novel genes that function under amenable conditions for microbial mesophilic growth in conventional hosts such as Escherichia coli or Saccharomyces cerevisiae. 31 putative GGRs were selected to test for potential reductase activity in vitro on farnesyl pyrophosphate, geranylgeranyl pyrophosphate, farnesol (FOH), and geranylgeraniol (GGOH). We report the discovery of several novel GGRs exhibiting significant activity toward various polyprenyl substrates under mild conditions (i.e., pH 7.4, T = 37 °C), including the discovery of a novel bacterial GGR isolated from Streptomyces coelicolor. In addition, we uncover new mechanistic insights within several GGR variants, including GGR-mediated phosphatase activity toward polyprenyl pyrophosphates and the first demonstration of completely hydrogenated GGOH and FOH substrates.ConclusionThese collective results enhance the potential for metabolic engineers to manufacture a variety of isoprenoid-based biofuels, polymers, and chemical feedstocks in common microbial hosts such as E. coli or S. cerevisiae.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1342-2) contains supplementary material, which is available to authorized users.
Research on renewable biofuels produced by microorganisms has enjoyed considerable advances in academic and industrial settings. As the renewable ethanol market approaches maturity, the demand is rising for the commercialization of more energy-dense fuel targets. Many strategies implemented in recent years have considerably increased the diversity and number of fuel targets that can be produced by microorganisms. Moreover, strain optimization for some of these fuel targets has ultimately led to their production at industrial scale. In this review, the recent metabolic engineering approaches for augmenting biofuel production derived from alcohols, isoprenoids, and fatty acids in several microorganisms are discussed. In addition, the successful commercialization ventures for each class of biofuel targets are discussed.
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