8 research outputs found
Rerouting Cellular Electron Flux To Increase the Rate of Biological Methane Production
Methanogens are anaerobic archaea that grow by producing methane, a gas that is both an efficient renewable fuel and a potent greenhouse gas. We observed that overexpression of the cytoplasmic heterodisulfide reductase enzyme HdrABC increased the rate of methane production from methanol by 30% without affecting the growth rate relative to the parent strain. Hdr enzymes are essential in all known methane-producing archaea. They function as the terminal oxidases in the methanogen electron transport system by reducing the coenzymeM(2-mercaptoethane sulfonate) and coenzyme B (7-mercaptoheptanoylthreonine sulfonate) heterodisulfide, CoM-S-S-CoB, to regenerate the thiol-coenzymes for reuse. In Methanosarcina acetivorans, HdrABC expression caused an increased rate of methanogenesis and a decrease in metabolic efficiency on methylotrophic substrates. When acetate was the sole carbon and energy source, neither deletion nor overexpression of HdrABC had an effect on growth or methane production rates. These results suggest that in cells grown on methylated substrates, the cell compensates for energy losses due to expression of HdrABC with an increased rate of substrate turnover and that HdrABC lacks the appropriate electron donor in acetate-grown cells
Rerouting Cellular Electron Flux To Increase the Rate of Biological Methane Production
Methanogens are anaerobic archaea that grow by producing methane, a gas that is both an efficient renewable fuel and a potent greenhouse gas. We observed that overexpression of the cytoplasmic heterodisulfide reductase enzyme HdrABC increased the rate of methane production from methanol by 30% without affecting the growth rate relative to the parent strain. Hdr enzymes are essential in all known methane-producing archaea. They function as the terminal oxidases in the methanogen electron transport system by reducing the coenzymeM(2-mercaptoethane sulfonate) and coenzyme B (7-mercaptoheptanoylthreonine sulfonate) heterodisulfide, CoM-S-S-CoB, to regenerate the thiol-coenzymes for reuse. In Methanosarcina acetivorans, HdrABC expression caused an increased rate of methanogenesis and a decrease in metabolic efficiency on methylotrophic substrates. When acetate was the sole carbon and energy source, neither deletion nor overexpression of HdrABC had an effect on growth or methane production rates. These results suggest that in cells grown on methylated substrates, the cell compensates for energy losses due to expression of HdrABC with an increased rate of substrate turnover and that HdrABC lacks the appropriate electron donor in acetate-grown cells
Interactions between Small Heat Shock Protein Subunits and Substrate in Small Heat Shock Protein-Substrate Complexes
Small heat shock proteins (sHSPs) are dynamic oligomeric
proteins that bind unfolding proteins and protect
them from irreversible aggregation. This binding results
in the formation of sHSP-substrate complexes from
which substrate can later be refolded. Interactions between
sHSP and substrate in sHSP-substrate complexes
and the mechanism by which substrate is transferred to
ATP-dependent chaperones for refolding are poorly defined.
We have established C-terminal affinity-tagged
sHSPs from a eukaryote (pea HSP18.1) and a prokaryote
(Synechocystis HSP16.6) as tools to investigate these issues.
We demonstrate that sHSP subunit exchange for
HSP18.1 and HSP16.6 is temperature-dependent and
rapid at the optimal growth temperature for the organism
of origin. Increasing the ratio of sHSP to substrate
during substrate denaturation decreased sHSP-substrate
complex size, and accordingly, addition of substrate
to pre-formed sHSP-substrate complexes increased
complex size. However, the size of pre-formed
sHSP-substrate complexes could not be reduced by addition
of more sHSP, and substrate could not be observed
to transfer to added sHSP, although added sHSP
subunits continued to exchange with subunits in sHSPsubstrate
complexes. Thus, although some number of
sHSP subunits within complexes remain dynamic and
may be important for complex structure/solubility, association
of substrate with the sHSP does not appear to be
similarly dynamic. These observations are consistent
with a model in which ATP-dependent chaperones associate
directly with sHSP-bound substrate to initiate
refolding
Computer-assisted Docking of Flavodoxin with the ATP:Co(I)rrinoid Adenosyltransferase (CobA) Enzyme Reveals Residues Critical for Protein-Protein Interactions but Not for Catalysis
The activity of the housekeeping ATP:co(I)rrinoid adenosyltransferase
(CobA) enzyme of Salmonella enterica sv. Typhimurium
is required to adenosylate de novo biosynthetic intermediates
of adenosylcobalamin and to salvage incomplete and complete
corrinoids from the environment of this bacterium. In vitro,
reduced flavodoxin (FldA) provides an electron to generate the
co(I)rrinoid substrate in the CobA active site. To understand how
CobAand FldA interact, a computer model of aCobA-FldA complex
was generated. This model was used to guide the introduction of
mutations into CobA using site-directed mutagenesis and the synthesis
of a peptide mimic of FldA. Residues Arg-9 and Arg-165 of
CobA were critical for FldA-dependent adenosylation but were catalytically
as competent as the wild-type protein when cob(I)alamin
was provided as substrate. These results indicate that Arg-9 and
Arg-165 are important for CobA_FldA docking but not to catalysis.
A truncation of the 9-amino acid N-terminal helix of CobA reduced
its FldA-dependent cobalamin adenosyltransferase activity by
97.4%. The same protein, however, had a 4-fold higher specific
activity than the native enzyme when cob(I)alamin was generated
chemically in situ
The Identity of Proteins Associated with a Small Heat Shock Protein during Heat Stress \u3ci\u3ein Vivo\u3c/i\u3e Indicates That These Chaperones Protect a Wide Range of Cellular Functions
The small heat shock proteins (sHSPs) are a ubiquitous
class of ATP-independent chaperones believed to
prevent irreversible protein aggregation and to facilitate
subsequent protein renaturation in cooperation
with ATP-dependent chaperones. Although sHSP chaperone
activity has been studied extensively in vitro, understanding
the mechanism of sHSP function requires
identification of proteins that are sHSP substrates in
vivo. We have used both immunoprecipitation and affinity
chromatography to recover 42 proteins that specifically
interact with Synechocystis Hsp16.6 in vivo during
heat treatment. These proteins can all be released from
Hsp16.6 by the ATP-dependent activity of DnaK and cochaperones
and are heat-labile. Thirteen of the putative
substrate proteins were identified by mass spectrometry
and reveal the potential for sHSPs to protect cellular
functions as diverse as transcription, translation, cell
signaling, and secondary metabolism. One of the putative
substrates, serine esterase, was purified and tested
directly for interaction with purified Hsp16.6. Hsp16.6
effectively formed soluble complexes with serine esterase
in a heat-dependent fashion, thereby preventing formation
of insoluble serine esterase aggregates. These
data offer critical insights into the characteristics of
native sHSP substrates and extend and provide in vivo
support for the chaperone model of sHSP function
The Identity of Proteins Associated with a Small Heat Shock Protein during Heat Stress \u3ci\u3ein Vivo\u3c/i\u3e Indicates That These Chaperones Protect a Wide Range of Cellular Functions
The small heat shock proteins (sHSPs) are a ubiquitous
class of ATP-independent chaperones believed to
prevent irreversible protein aggregation and to facilitate
subsequent protein renaturation in cooperation
with ATP-dependent chaperones. Although sHSP chaperone
activity has been studied extensively in vitro, understanding
the mechanism of sHSP function requires
identification of proteins that are sHSP substrates in
vivo. We have used both immunoprecipitation and affinity
chromatography to recover 42 proteins that specifically
interact with Synechocystis Hsp16.6 in vivo during
heat treatment. These proteins can all be released from
Hsp16.6 by the ATP-dependent activity of DnaK and cochaperones
and are heat-labile. Thirteen of the putative
substrate proteins were identified by mass spectrometry
and reveal the potential for sHSPs to protect cellular
functions as diverse as transcription, translation, cell
signaling, and secondary metabolism. One of the putative
substrates, serine esterase, was purified and tested
directly for interaction with purified Hsp16.6. Hsp16.6
effectively formed soluble complexes with serine esterase
in a heat-dependent fashion, thereby preventing formation
of insoluble serine esterase aggregates. These
data offer critical insights into the characteristics of
native sHSP substrates and extend and provide in vivo
support for the chaperone model of sHSP function
Purification and Initial Biochemical Characterization of ATP:Cob(I)alamin Adenosyltransferase (EutT) Enzyme of \u3ci\u3eSalmonella enterica\u3c/i\u3e
ATP:cob(I)alamin adenosyltransferase (EutT) of Salmonella
enterica was overproduced and enriched to ~70% homogeneity,
and its basic kinetic parameters were determined. Abundant
amounts of EutT protein were produced, but all of it remained
insoluble. Soluble active EutT protein (~70% homogeneous) was
obtained after treatment with detergent. Under conditions in which
cobalamin (Cbl) was saturating, Km(ATP) = 10 µM, kcat = 0.03 s-1,
and Vmax = 54.5 nM min-1. Similarly, under conditions in which
MgATPwas saturating,Km(Cbl) = 4.1µM, kcat = 0.06 s-1, andVmax=
105 nM min-1. Unlike other ATP:co(I)rrinoid adenosyltransferases
in the cell (i.e. CobA and PduO), EutT activity was \u3e50-fold higher
with ATP versus GTP, and EutT retained 80% of its activity with
ADP substituted for ATP and was completely inactive with AMP as
substrate, indicating that the enzyme requires the β-phosphate
group of the nucleotide substrate. The data suggest that the amino
group of adenine might play a role in nucleotide recognition and/or
binding. Unlike the housekeeping CobA enzyme, EutT was not
inhibited by inorganic tripolyphosphate (PPPi). Results from 31P
NMR spectroscopy studies identified PPi and Pi as by-products of
the EutT reaction. In the absence of Cbl, EutT cleaved ATP into
adenosine and PPPi, suggesting that PPPi is broken down into PPi
and Pi. Electron transfer protein partners for EutT were not
encoded by the eut operon. EutT-dependent activity was detected in
cell-free extracts of cobA strains enriched for EutT when FMN and
NADH were used to reduce cob(III)alamin to cob(I)alamin
The ATP:Co(I)rrinoid Adenosyltransferase (CobA) Enzyme of \u3ci\u3eSalmonella enterica\u3c/i\u3e Requires the 2’-OH Group of ATP for Function and Yields Inorganic Triphosphate as Its Reaction Byproduct
The specificity of the ATP:corrinoid adenosyltransferase
(CobA) enzyme of Salmonella enterica serovar
Typhimurium LT2 for its nucleotide substrate was
tested using ATP analogs and alternative nucleotide donors.
The enzyme showed broad specificity for the nucleotide
base and required the 2’-OH group of the ribosyl
moiety of ATP for activity. 31P NMR spectroscopy
was used to identify inorganic triphosphate (PPPi) as
the byproduct of the reaction catalyzed by the CobA
enzyme. Cleavage of triphosphate into pyrophosphate
and orthophosphate did not occur, indicating that
triphosphate cleavage was not required for release of
the adenosylcorrinoid product. Triphosphate was a
strong inhibitor of the reaction, with 85% of CobA activity
lost when the ATP/PPPi ratio present in the reaction
mixture was 1:2.5