From the Department of Microbiology, University of Illinois, Urbana, Illinois 61801
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We discovered that
Methanobacterium thermoautotrophicum strain H possessed
pyruvate carboxylase (PYC), and this biotin prototroph required
exogenously supplied biotin to exhibit detectable amounts of PYC
activity. The enzyme was highly labile and was stabilized by 10%
inositol in buffers to an extent that allowed purification to
homogeneity and characterization. The purified enzyme was absolutely dependent on ATP, Mg2+ (or Mn2+ or
Co2+), pyruvate, and bicarbonate for activity;
phosphoenolpyruvate could not replace pyruvate, and acetyl-CoA was not
required. The enzyme was inhibited by ADP and
-ketoglutarate but not
by aspartate or glutamate. ATP was inhibitory at high concentrations.
The enzyme, unlike other PYCs, exhibited nonlinear kinetics with
respect to bicarbonate and was inhibited by excess Mg2+,
Mn2+, or Co2+. The 540-kDa enzyme of
A4B4 composition contained a non-biotinylated 52-kDa subunit (PYCA) and a 75-kDa biotinylated subunit (PYCB). The
pycB gene was probably monocistronic and followed by a
putative gene of a DNA-binding protein on the opposite strand. The
pycA was about 727 kilobase pairs away from
pycB on the chromosome and was probably co-transcribed with
the biotin ligase gene (birA). PYCA and PYCB showed
substantial sequence identities (33-62%) to, respectively, the biotin
carboxylase and biotin carboxyl carrier + carboxyltransferase domains
or subunits of known biotin-dependent carboxylases/decarboxylases. We
discovered that PYCB and probably the equivalent domains or subunits of
all biotin-dependent carboxylases harbored the
serine/threonine dehydratase types of pyridoxal-phosphate attachment
site. Our results and the existence of an alternative oxaloacetate
synthesizing enzyme phosphoenolpyruvate carboxylase in M. thermoautotrophicum strain
H (Kenealy, W. R., and Zeikus, J. G. (1982) FEMS Microbiol. Lett. 14, 7-10) raise
several questions for future investigations.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Pyruvate carboxylase (PYC)1 catalyzes ATP-dependent carboxylation of pyruvate to generate oxaloacetate. The other possible routes for oxaloacetate synthesis are carboxylation of phosphoenolpyruvate (PEP) by PEP carboxylase (PPC) and PEP carboxytransphosphorylase, splitting of citrate by citrate lyase and ATP-citrate lyase, and the reversal of the PEP carboxykinase reaction (1). Of these enzymes, PYC and PPC are more commonly employed enzymes for generating oxaloacetate (1-3), and only rarely do they co-exist in a given organism (2, 4-6). A similar pattern has been found in methanogenic archaea. Methanococcus possesses PYC but not PPC (7). Enzyme assays and isotope labeling studies suggest that Methanobacterium possesses PPC and is devoid of PYC and PEP carboxytransphosphorylase activities (8, 9). In Methanosarcina, PPC is absent (10), and the PYC activity has yet to be demonstrated.
In mammals and yeast, PYC activity is responsible for replenishing
oxaloacetate for continued operation of the tricarboxylic acid cycle
(3). In the absence of this anaplerotic function, consumption in cell
material biosynthesis depletes the oxaloacetate pool. PYC, in
conjunction with PEP carboxykinase, also provides PEP that is needed
for gluconeogenesis, since the pyruvate kinase reaction of the
glycolytic pathway is irreversible (1-3). PYC is also present in
plants, where its role has yet to be established (11). In
Escherichia coli, PPC provides oxaloacetate during growth on
glucose (1). Since E. coli does not possess PYC, during
growth on acetate it employs the glyoxalate cycle to generate oxaloacetate for gluconeogenesis. Depending on the growth conditions, Pseudomonas citronellolis uses either PYC or PPC as the
anaplerotic enzyme (5). In methanogens, PYC and PPC activities serve
anabolic functions. In Methanococcus,
Methanobacterium, and Methanospirillum, oxaloacetate is the starting point of an incomplete reductive tricarboxylic acid cycle that terminates at -ketoglutarate and provides several precursors for cell material and coenzyme biosynthesis (7, 12-14). In Methanosarcina, oxaloacetate initiates an
incomplete oxidative tricarboxylic acid cycle to generate
-ketoglutarate (10).
PYC belongs to a large family of biotinylated enzymes that carry out
carboxyl-group transfer in a variety of reactions (3, 15). These
enzymes use biotin as a "swinging arm" to transfer a
COO
group between active sites and show strong
conservation at the amino acid sequence level (15, 16). They also carry
out analogous partial reactions, which for PYCs are as indicated in
Reactions 1 and 2.
![]() |
![]() |
![]() |
![]() |
![]() |
The following nomenclatures have been used for describing the
individual polypeptides (and corresponding genes) of M. thermoautotrophicum H pyruvate carboxylase: PYCA
(pycA) for the polypeptide possessing the ATP binding motif
(with A representing ATP) and PYCB (pycB) for the
biotinylated polypeptide (B for biotin).
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Organisms and Culture Conditions--
M.
thermoautotrophicum strain H (19) was grown in tubes, bottles,
and in a 14-liter stainless steel fermentor (Microferm, New Brunswick
Scientific, New Brunswick, NJ) in Medium 1 of Balch and Wolfe (20) as
described previously (20, 21). For studying the effect of medium
compositions on the expression of PYC, the growth medium was modified
as described under "Results." The cells grown in the fermentor were
harvested by using a continuous flow centrifuge, frozen in liquid
nitrogen, and stored at
70 °C (21). E. coli DH5
was
used as the cloning host, and this strain was grown in LB medium at
37 °C. Wherever needed, the LB medium was supplemented with
ampicillin (100 µg/ml), 5-bromo-4-chloro-3-indolyl
-D-galactoside (40 µg/ml), and
isopropyl-
-D-thiogalactopyranoside (100 µM).
Purification of Pyruvate Carboxylase-- A lysis buffer stock of the following composition was used to resuspend the frozen cells: 100 mM Tris base, 2 M KCl, 10% inositol, and 10 mM MgCl2, pH 8 (adjusted with HCl). Dithiothreitol (DTT) was added to the cell suspension from a freshly prepared stock. The final composition of the cell suspension was as follows: 50 mM Tris buffer, 1 M KCl, 5% inositol, 5 mM MgCl2, 4 mM DTT, and ~0.5 g wet cells/ml. The cells were disrupted by three passages through a French pressure cell at 1240 atm. The broken cell slurry was centrifuged at 17,000 × g for 30 min at 4 °C. The resulting supernatant was recentrifuged at 100,000 × g for 40 min at 4 °C. The pellet from this stage was discarded, and the supernatant was used for enzyme purification.
From the cell extract, pyruvate carboxylase was purified to homogeneity by single step affinity chromatography using a monomerized avidin-Sepharose column. This affinity matrix was prepared according to previously published protocols (22). Before use, the matrix was regenerated by washing with 0.1 M glycine-HCl at pH 2. The regenerated column was washed with 20 mM potassium phosphate buffer, pH 7, until the pH of the effluent was 7, and then was equilibrated with the column buffer of the following composition: 50 mM Tris, 1 M KCl, 10% inositol, 5 mM MgCl2, 2 mM DTT, pH 8 (adjusted with HCl). The cell extract was diluted with an equal volume of column buffer and, following the protocols of Purcell and Wallace (23), was supplemented with ATP, KHCO3, and sodium pyruvate to the final concentrations of 3, 10, and 10 mM, respectively, to improve accessibility to protein-bound bio-tin. The diluted cell extract was loaded onto the affinity column at a flow rate of ~0.75 cm/min either under gravity or by using a Tris® peristaltic pump (Isco, Inc., Lincoln, NE). The column bed was then washed with 10 bed volumes of column buffer to remove unbound material. Pyruvate carboxylase bound to the matrix very tightly and was eluted with 2 bed volumes of 1 mM D-biotin in column buffer.Assays and Data Analysis--
Protein was assayed
according to Bradford (24) using the dye reagent purchased
from Pierce. Pyruvate carboxylase was assayed in the direction of
oxaloacetate formation by coupling the reaction with malate
dehydrogenase. The oxidation of NADH in the malate dehydrogenase
reaction was followed spectrophotometrically at 340 nm. Unless
mentioned otherwise, the assay mixture contained 50-100 mM
Tris-HCl buffer at pH 8, 4 mM MgCl2, 400 mM KCl, 50 mM sodium pyruvate, 50 mM KHCO3, 4 mM ATP, 0.2 mM NADH, and 1 unit of malate dehydrogenase from
Thermus flavus (Sigma). The assays were initiated by the
addition of 10-100 µl of pyruvate carboxylase preparations to 1 ml
of pre-warmed assay mixture. For pH studies the Tris-HCl buffer was
replaced with buffers containing 60 mM each of MES, Tris,
and glacial acetic acid and adjusted to the desired pH (6-9.5) with
HCl or NaOH; the values calculated (25) for the ionic strengths of
these buffers were between 0.06 and 0.09 units. All initial rate data
were analyzed by using the KinetAsyst program version 1.01 (Intellikinetics, State College, PA), except those for bicarbonate,
which were fitted to 2/1 function (v =
Vm(S2 + DS)/(S2 + BS + C) where B, C, and
D are constants and Km = 0.5B D + {(0.5B
D)2 + C}1/2 (26)).
Gel Electrophoresis and Western Blot Analysis-- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed with 12.5% polyacrylamide slab gels according to Laemmli (27). For nondenaturing PAGE, 5% polyacrylamide gels and a variety of gel and electrode buffers were used. The best results were obtained when 100 mM phosphate buffer, pH 7.1, with 10% inositol and 10 mM MgCl2 served as both the gel buffer and the electrode buffer (see "Results").
For Western blot analysis, the protein samples were electrophoresed and electroblotted onto Immobilon-P transfer membranes (Millipore Corp., Bedford, MA). The membranes with blotted polypeptides were washed with phosphate-buffered saline (PBS) (10 mM sodium phosphate buffer, pH 7.2, 150 mM NaCl) and then blocked with 3% bovine serum albumin in PBS for 2 h. The blocked membranes were washed three times with PBS and shaken for 30 min at 4 °C while immersed in 0.01 mg/liter alkaline phosphatase-conjugated avidin (Sigma) in PBS. The membranes were then washed with PBS, equilibrated in a veronal buffer (32 mM veronal, 30 mM sodium acetate, 120 mM NaCl, pH 9.6) for 5 min, and developed with nitro blue tetrazolium (350 µg/ml) and bromochloroindoyl phosphate (175 µg/ml) in the veronal buffer. The use of VECTASTAIN ABC-AP reagent (Vector Laboratories, Burlingame, CA) in place of alkaline phosphatase-conjugated avidin gave very high background, with the majority of the protein bands reacting with avidin.Gel Filtration Chromatography--
An HR 10/30 Superose-6 column
and a fast protein liquid chromatography system (Pharmacia Biotech
Inc.) was used for this purpose. The mobile phase was 50 mM
Tris-HCl buffer, pH 7.5, 100 mM KCl, and 5% glycerol, and
the flow rate was 0.5 ml/min. The molecular mass standards in kDa were
thyroglobulin (669), apoferritin (443), -amylase (200), alcohol
dehydrogenase (150), bovine serum albumin (60), carbonic anhydrase
(29), and vitamin B12 (1.357). Before applying the purified
enzyme, the column was equilibrated with the mobile phase supplemented
with 1 mM DTT and 5 mM MgCl2.
The eluted proteins were detected by their absorbance at 280 nm.
Determination of the Amino-terminal Amino Acid Sequence-- The purified pyruvate carboxylase was electrophoresed in a 12.5% polyacrylamide slab gel under denaturing conditions, and the separated subunits were electroblotted onto a Pro-Blott membrane (Applied Biosystems, Foster City, CA) according to manufacturer protocols using Tris/glycine/methanol as the blotting buffer. The pieces of membrane containing individual subunits, as visualized by staining with Coomassie Brilliant Blue, were used for sequencing by Edman degradation at the University of Illinois Genetic Engineering Facility.
DNA Methods--
Generally, all manipulations were performed
according to standard methods (28). Chromosomal DNA from M. thermoautotrophicum strain H was isolated according to
Mukhopadhyay et al. (29). The plasmid pBluescript II
SK+ (Stratagene, La Jolla, CA) was used as the cloning
vector. Plasmids from E. coli cell lysates were purified by
using Qiagen Tips and reagents from Qiagen (Chatsworth, CA). DNA
fragments of interest were purified from agarose gels by using either
Quiaquick columns (Qiagen) or by digestion with
-agarase (New
England Biolabs, Inc., Beverly, MA) or AgarACE (Promega Corp., Madison,
WI). Oligonucleotides that were used as hybridization probes were
labeled at the 3'-end with digoxigenin-dideoxy-UTP using a kit (Genius
3) from Boehringer Mannheim according to manufacturer instructions. The
pre-hybridization and hybridization were conducted at 55 °C and
post-hybridization washes were at 24 °C. The hybridizing bands were
detected by using alkaline phosphatase-conjugated anti-digoxigenin
antibody (Boehringer Mannheim) and the colorimetric substrates nitro
blue tetrazolium and bromochloroindoyl phosphate. DNA sequencing was
performed at the University of Iowa DNA Facility (Iowa City, IA) using
an automated sequencer. The first round of sequencing was with plasmids pBM1 and pBM2 using the primers based on the vector sequences. Additional sequencing was from pBM1 using primers based on the accumulated sequences. Both strands were sequenced.
DNA and Protein Sequence Analysis-- The DNA sequences were assembled, aligned, and analyzed using the DNA Star (DNASTAR Inc., Madison, WI) program. Data base searches were performed using the BLAST program (30) of National Center for Biotechnology Information or NCBI (National Institutes of Health). Multiple protein sequence alignments were carried out using the program ClustalW. The primary structures of polypeptides were analyzed by using various programs as indicated.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Purification and Molecular Properties of Pyruvate Carboxylase from
M. thermoautotrophicum Strain H--
Most of the pyruvate
carboxylase activity was found in the 100,000 × g
supernatant of the cell extract containing 1 M KCl. Thus,
it is a soluble and hydrophilic protein. The enzyme bound tightly to
monomerized avidin-Sepharose and was eluted from this matrix with 1 mM D-biotin as a single sharp peak. As judged
by nondenaturing PAGE patterns, the product was homogeneous (Fig. 1). The presence of 1 M KCl
in the buffer was critical for obtaining a good yield and purity, which
was typically 1-1.5 mg of enzyme/200 g of wet cell paste.
Inositol was required for maintaining activity. We were unable to
recover active fractions when glycerol was used in place of
inositol.
|
|
Catalytic Properties of M. thermoautotrophicum Strain H Pyruvate
Carboxylase--
The activity of the purified enzyme was strictly
dependent on the presence of ATP, pyruvate, bicarbonate, and
Mg2+ (supplied either as MgCl2,
MgSO4, or Mg-ATP). Pyruvate could not be replaced with PEP.
GTP, CTP, UTP, ITP, or ADP did not substitute for ATP. Incubation of
purified enzyme with excess avidin completely inhibited its activity
(Table I), establishing the typical
dependence of pyruvate carboxylase activity on protein-bound biotin for
the Methanobacterium enzyme. This inactivation did not occur
if avidin was incubated with excess biotin prior to its addition to the enzyme. Addition of biotin after avidin had acted on the enzyme restored only a very minor portion of the original activity. The purified enzyme exhibited maximum activity at pH 8. Measurable activities of the enzyme were seen throughout the range of
30-80 °C, and the maximum specific activity was recorded at
60 °C.
|
|
Cloning and Sequencing of the Gene for the Biotinylated Subunit of
PYC--
Southern blot analysis of EcoRI-digested M. thermoautotrophicum H DNA with a 100% degenerate (at all
wobble positions) oligonucleotide 5' RAA NGC NGT YTC NAC NAC YTT DAT
NCC YTT 3', corresponding to the NH2-terminal amino acid
sequence (residue 2-11) of the 75-kDa subunit of purified PYC, showed
a hybridization signal at 3-4-kb position. Accordingly, using
pBluescript II SK+ as the vector, a limited library of
3-5-kb EcoRI fragments of M. thermoautotrophicum
H genomic DNA was constructed in E. coli DH5
. This
library was screened by colony hybridization using the degenerate
oligonucleotide as the probe. Ten of these colonies showed positive
hybridization signals, and the corresponding recombinant plasmids had
inserts of size ~3.5 kb. One of these strains, bearing the
recombinant plasmid designated pBM1, was preserved. Fig.
4A shows the restriction map
of the M. thermoautotrophicum
H DNA insert in pBM1. The
2.1-kb EcoRI-XhoI fragment of this insert was
subcloned into pBluescript II SK+ giving the plasmid pBM2
(Fig. 4A). Fig. 4B shows the DNA sequence of the
entire clone in pBM1 and the deduced amino acid sequence of the
biotinylated subunit of PYC.
|
DNA Sequence Analysis--
The DNA sequence shown in Fig.
4B harbored three open reading frames of significant
lengths. From comparison with the determined NH2-terminal
amino acid sequence as reported above, the largest of these open
reading frames was identified as the gene for the biotinylated or B
subunit of pyruvate carboxylase of M. thermoautotrophicum strain H (Fig. 4B). This gene was designated as
pycB and the corresponding gene product as PYCB. For the
pycB gene, ATG was the start codon and TAA was the stop
codon (Fig. 4B). The pycB gene sequence was 54 mol % G + C, and this value was comparable to the overall mol % G + C content (48%) of M. thermoautotrophicum
H genome (31).
The initiation codon of the pycB gene was preceded by the
sequence AGAGG (position
11 to
7), which could serve as a
ribosome-binding site. The available sequence upstream of pycB did not harbor any open reading frame of significant
length but several AT-rich stretches resembling TATA box component of methanogen promoters (32). Several oligo(dT) sequences that might
provide transcription termination signals (33) were found downstream of
the pycB gene (underlined sequences in Fig.
4B). This downstream region also contained an inverted
repeat CAtAAaATAAAAaccttcTTTTATaTTcTG that included last 10 bases of
the pycB gene including the termination codon and could form
a stem and loop structure.
Deduced Properties of PYCB Polypeptide--
A comparison of the
deduced PYCB sequence with the determined NH2-terminal
amino acid sequence of the 75-kDa subunit of purified enzyme suggested
that the initiator methionine was retained in the matured PYCB peptide.
The calculated molecular mass of the PYCB peptide was 63961 daltons,
about 11-kDa smaller than the value obtained from SDS-PAGE with
purified enzyme. The theoretical pI of the protein was 4.66 and the
aliphatic index was 90.25. The net charge of the protein at pH 7 and 9, as calculated by using the ISOELECTRIC program of the GCG package
(Genetic Computer Group Inc., Madison, WI), were 39.87 and
50.16,
respectively. An analysis by using the SOUSI program (Mitaku
Laboratory, University of Tokyo Agricultural and Technology, Tokyo)
predicted that PYC was devoid of potential transmembrane segments and
was a soluble protein with a hydrophobicity of
0.286. A PROSITE
search (University of Geneva) revealed that the sequence
EALECDSVAIK174DMAG (residues 164-178) of PYCB could
represent a serine/threonine dehydratase type pyridoxal-phosphate
attachment site (accession number PS00165).
|
|
The pycA Gene and Deduced Properties of PYCA
Polypeptide--
Since a comparison of the NH2-terminal
sequence of the 52-kDa subunit to the available sequences in the data
base showed high degrees of similarities to bacterial biotin
carboxylases and the putative biotin carboxylase of M. jannaschii (34), this subunit was assumed to be the biotin
carboxylase unit of PYC. Since the biotin carboxylases bind ATP, this
subunit was named PYCA. A search in the recently available unpublished
and tentative sequence of the entire genome of M. thermoautotrophicum strain H3 revealed that the
pycA gene was 727 kilobase pairs or about half a genome away
from pycB and was immediately (4 bp downstream) followed by
a putative biotin ligase (birA) gene. Analysis of the
sequences upstream of pycA and downstream of putative
birA suggested that these two genes are probably
co-transcribed. The calculated molecular mass of the PYCA peptide was
54,656 daltons and the aliphatic index was 86.07. The theoretical pI of
the protein was 6.15 and the net charges at pH 7 and 9 were
2.72 and
9.72, respectively, making PYCA a neutral polypeptide at
physiological pH. PYCA was predicted to be a soluble protein with a
hydrophobicity of
0.286. The PYCA sequence was found to be highly
similar to the NH2-terminal half of the
4
PYCs from eukaryotes (human (39) and yeast (16); average identity,
45%) and bacteria (R. etli (41), 43% identity) and to the
entire sequences of biotin carboxylase subunits of several
multi-subunit biotin-dependent carboxylases (human
propionyl-CoA corboxylase
subunit or PCCA (46), 48% identity;
M. jannaschii biotin carboxylase (34), 62% identity). A
multiple alignment of these sequences (Fig.
7) revealed that the ATP binding motif
and other sequence features, which are usually associated with the
biotin carboxylases, were present in PYCA; note that we renamed the
M. jannaschii BC as M. jannaschii PYCA for the
reasons given under "Discussion." The sequence GGGGIGMRAV of PYCA
(residues 162-171) matched the consensus GGGGXGMRUU (U = A, I, L, V) sequence that is implicated in binding ATP by biotin carboxylases (39, 41, 47). This consensus sequence is similar to the
"P loop" motif GXXXXGK(TS) that is involved in binding ATP or the nucleotide portion of nicotinamides in several ATP- or
nicotinamide-dependent enzymes (48, 49). The
Cys229 of PYCA, which was located 62 residues downstream of
the last Gly of the putative ATP binding motif, was also highly
conserved in all biotin carboxylases. This Cys residue is commonly
found as a part of the sequence DCS (ECS at location 228-230 in PYCA) and is believed to be involved in the CO2 fixation reaction
by biotin-dependent enzymes (47). From x-ray
crystallographic studies Waldrop et al. (50) showed that in
the biotin carboxylase subunit of E. coli acetyl-CoA
carboxylase, the residues His209-Glu211,
His236-Glu241, Glu276,
Ile287-Glu296, and Arg338 probably
form part of the active site pocket, and some of these residues
(Lys238, Arg292, Gln294,
Glu296, and Arg338) might bind a hydrogen
phosphate ion. In the PYCA sequence (Fig. 7) the corresponding residues
were probably His208-Glu210,
His235-Glu240, Glu275,
Leu286-Glu295, and Arg337. Also the
NH2-terminal region (residue 1-103) of PYCA was found to
be 50% identical to that of E. coli biotin carboxylase, and this region in the latter protein has been found by Waldrop et al. (50) to adopt a dinucleotide binding motif making it a
possible candidate for binding ATP.
|
Conditions for Expression of PYC in M. thermoautotrophicum Strain
H--
For routine purification of PYC, M. thermoautotrophicum strain
H cells grown in Medium 1 of Balch
and Wolfe (20) were used as the starting material. Despite repeated
attempts, we were unable to recover active PYC preparations from
autotrophically grown cells, and the corresponding affinity column
fractions from wash with 1 mM D-biotin did not
show any protein band in SDS-PAGE or any avidin-reacting band in
Western blots. To explore the reason for this observation, we performed
Western blot analysis with extracts of cells grown in the following
three media: Medium 1, autotrophic or minimal medium (Medium 1 without
acetate, yeast extract, tryptone, and vitamins), and autotrophic medium
supplemented with D-biotin to a final concentration of 100 µM. The results from these experiments are shown in Fig.
8. The autotrophically grown cells were
either devoid of this protein band or possessed it below the detection
limit of the system employed. A 75-kDa avidin reacting band was seen
when the growth medium contained either D-biotin or yeast
extract + acetate + tryptone + vitamins.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We discovered that the pyruvate carboxylase (PYC) activity was
present in M. thermoautotrophicum strain H when complex
components and vitamin solutions that contained biotin or biotin itself
were added to the growth medium. These results explain the failures by
previous workers to find PYC in cells grown under autotrophic conditions (9). A similar situation probably exists with the closely
related organism M. thermoautotrophicum Marburg (31), which
has been reported to be devoid of PYC activity (8). The presence of
both PYC and PPC activities and regulation of PYC activity by
exogenously supplied biotin in the methanoarchaeon M. thermoautotrophicum strain
H raise several questions. These aspects as well as the characteristics of the purified enzyme are
discussed below.
The bacterial and eukaryotic PYCs are homotetramers (4)
of 110-130-kDa subunits (3, 41, 51) with PYC of P. citronellolis (52) and probably Azobacter vinelandii
PYC (6) being the only known exceptions. The P. citronellolis PYC is of
4
4
structure, where the biotinylated
subunits (65 kDa) are on the
outside of the molecule and surround the 54-kDa
subunits that form
the core (52). Also, the
4
4 PYCs, unlike
the
4 enzymes, do not require acetyl-CoA for activity or
stability and are insensitive to tricarboxylic acid cycle members or
related metabolites. Thus, in respect to the quaternary structure and
the requirement of or response to effectors, the PYC of M. thermoautotrophicum
H was found to be a typical
4
4-type enzyme, except it was very mildly
inhibited by
-ketoglutarate. Our analysis of published data (34)
suggested that M. jannaschii most likely possesses a
M. thermoautotrophicum-type PYC (see below). Similar to all other PYCs, the PYC of M. thermoautotrophicum was absolutely
dependent on ATP and was inhibited by ADP.
Our preliminary analysis revealed that the M. thermoautotrophicum PYC possessed complex kinetic properties. Unlike other PYCs (6, 51, 53, 54), this enzyme showed negative cooperativity with respect to bicarbonate and followed Henri-Michaelis-Menten relationship with respect to pyruvate. Earlier studies with pyruvate carboxylases showed that the inhibition by ATP could be relieved by excess Mg2+, Mn2+, or Co2+ (6, 53-55). For the M. thermoautotrophicum PYC, an increase in Mg2+ concentration beyond that of ATP increased the apparent affinity for ATP but decreased the apparent Vm. The inhibition of methanogen PYC by free Mg2+ was in congruence with the observation that Mn2+ or Co2+ when supplied in place of Mg2+ supported PYC activity but when present along with sufficient amount of Mg2+ inhibited the reaction severely. On the other hand, activation of desalted enzyme preparations by Mg2+ suggested that binding of this ion to the protein was necessary for attaining a catalytically active configuration. A detailed study to fully understand the basis of these unique properties of the M. thermoautotrophicum enzyme is underway.
Since the PYCB peptide would be acidic at electrophoresis pH
(calculated value of the charge, 50), the SDS-PAGE-derived molecular weight value was expected to be lower than the sequence-derived value.
But our observation was the opposite. It was also difficult to explain
how a biotinylated band of 67 kDa could arise from the cleavage of
75-kDa subunit retaining the same NH2-terminal sequence,
because a cleavage at the COOH terminus that would retain the
biotinylation site AMKM would account for a mass change of only 3.6 kb.
The regulation of PYC activity in M. thermoautotrophicum
H by added biotin was intriguing, since the organism can synthesize biotin on its own (17). It seems that the elevation of intracellular biotin level beyond what can be maintained from biosynthesis was necessary for exhibiting detectable levels of PYC, and one or more of
the following reasons could account for this requirement. 1) Higher
biotin level was necessary for the (over)expression of PYC subunits
and/or the biotinylating enzyme biotin ligase. 2) The biotinylation of
PYC required higher biotin levels due to relatively low affinity of
ligase for biotin; the low affinity was either an intrinsic property of
the ligase or was imposed by a modulator. Regulation of activity for
biotin-dependent enzymes in a biotin-producing organism by
exogenously added biotin has been observed only rarely. In E. coli the only biotinylated enzyme is acetyl-CoA carboxylase (ACC)
and its level is not influenced by the addition of biotin to the growth
medium. Rather, in this organism the level of expression of ACC
determines the biotin biosynthesis rate (56, 57). In most organisms the
expression of PYC is also not influenced by exogenous biotin. However,
in B. stearothermophilus, Bacillus coagulans,
Brevibacterium latofermentum, and R. etli PYC
activity is found only if biotin is added to the growth medium,
although their ACC activities do not show such a dependence (4,
58-60). When grown in the absence of added biotin, B. coagulans synthesizes apo-PYC and biotin ligase but cannot
biotinylate the apoenzyme (58); the corresponding extract can produce
active holoenzyme if excess biotin is added. It is not known whether
this observation is due to a general purpose ligase that shows low
affinity for biotin while ligating biotin to PYC, but not for
biotinylating ACC, or due to the low affinity of a PYC-specific
ligase.
Only on rare occasions both PPC and PYC are found in a given organism,
and wherever they co-exist their activities are regulated differently.
Subculturing in minimal medium with succinate and biotin increases PYC
activity in R. etli, but its PPC activity remains unchanged
(4). P. citronellolis possesses both of PPC and PYC (5, 61).
In this organism PPC is constitutive, but its activity is modulated
through inhibition by aspartate and activation by ADP and acetyl-CoA
(5). On the other hand its PYC in insensitive to these modulators, but
both subunits of the enzyme are induced when a pyruvate-generating
carbon source is in use and are repressed if a carbon source that
converts readily to OAA is supplied (5, 61). A preliminary report on
M. thermoautotrophicum PPC shows that, similar to the PYC of
this organism, this enzyme is neither activated nor inhibited by
acetyl-CoA (9). Thus, our current knowledge is insufficient to predict
if and how M. thermoautotrophicum H controls the
expression and activity of PPC and PYC in response to physiological
conditions.
We propose to rename the putative biotin carboxylase (BC) and
oxaloacetate decarboxylase subunit (OAD
) of M. jannaschii (accession number, U67563) (34), respectively, as the A
and B subunits of pyruvate carboxylase. The current names were derived solely from comparative analyses of sequences in a whole genome sequencing project (34) and seemed justified in the light of observed
strong smilarities (see "Results"). However, for the following
reasons the proposed names describe the in vivo functions of
these polypeptides properly. First, the putative BC and OAD
polypeptides of M. jannaschii were, respectively, 62 and
61% identical to the M. thermoautotrophicum PYCA and PYCB.
Second, a search of the entire genome sequence of M. jannaschii (34) did not show the presence of another putative
pyruvate carboxylase or a putative PEP carboxylase. Methanococcus
maripaludis, an organism closely related to M. jannaschii, makes oxaloacetate using pyruvate carboxylase and is
devoid of phosphoenolpyruvate carboxylase (7). A similar situation is
expected for M. jannaschii.
The PYCA and BirA (biotin ligase) polypeptides would interact with PYCB for biotinylation and carboxylation reactions, respectively. In M. thermoautotrophicum the pycA and birA genes were found to be part of an operon located about half a genome away from pycB. It would be interesting to investigate how the regulation of expression of these polypeptides at two different chromosomal locations are coordinated to generate a functional oxaloacetate synthesizing system on demand. In contrast, in M. jannaschii the putative pycA and pycB genes along with an intervening open reading frame seem to form an operon, and the birA gene is about 422 kilobase pairs away from pycA (34). In E. coli the genes for the subunits of acetyl-CoA carboxylase are located at three different locations of the chromosome (62), and the system coordinating the regulation of expression for this enzyme is still unknown.
Our analysis of primary structures of two methanoarchaeal PYCs
demonstrated that the previously recorded high degrees of conservation in the primary structures of bacterial and eukaryotic
biotin-dependent carboxylases of diverse substrate
specificity extend into the domain of archaea, although the methanogen
enzyme possessed a unique structure. All biotin-dependent
carboxylases are composed of the following functional units: biotin
carrier or biotin carboxyl carrier, biotin carboxylase or BC
(possessing ATP-binding and CO2-fixation site), and
carboxyltransferase (possessing ketoacid- and metal ion-binding sites)
(15, 16, 38, 41). Several types of arrangements of these units
within the primary and quaternary structures of enzymes have been found
(Fig. 6 and references therein). The M. thermoautotrophicum PYC possessed two types of subunits and
therefore appeared to be similar to eukaryotic PCCs (Fig. 6). However,
a comparative analysis of primary structures (Figs. 5 and 7) showed
that it had a pattern of its own (Fig. 6). The larger subunit of
M. thermoautotrophicum PYC carried the putative biotin and
pyruvate-binding sites and therefore harbored both the biotin carboxyl
carrier and the carboxyltransferase domains, and the smaller subunit
had all of the sequence characteristics of a BC domain. Thus, if both
the eukaryotic PCCs and M. thermoautotrophicum PYC had
originated either from fusion of genes for the E. coli ACC-type enzymes or through splitting of the gene for
4-type PYCs, the reshuffling of gene segments occurred
differently for creating these two types of proteins.
Interestingly the M. thermoautotrophicum PYCB was found to harbor a putative serine/threonine dehydratases type pyridoxal-phosphate attachment site, and the corresponding region was highly conserved in other biotin-dependent carboxylases/decarboxylases (Fig. 5.). This site has not been identified before, and there is no report on the requirement of pyridoxal phosphate for the activity or regulation of biotin-dependent carboxylases/decarboxylases. We are currently investigating the role of pyridoxal phosphate in modulating activities of these enzymes.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Cindy Kreder for excellent technical assistance. We also thank Endang Purwantini, John Cronan, Jr., Dave Graham, and Carl Woese for helpful discussions. We thank Bryce V. Plapp and Endang Purwantini for help in kinetic analysis.
![]() |
Note Added in Proof |
---|
After submission of this manuscript, the
sequence of the complete genome of M. thermoautotrophicum
H was published (Smith, D. R., Doucette-Stamm, L. A.,
Deloughery, C., Lee, H., Dubois, J., Aldredge, T., Bashirzadeh, R.,
Blakely, D., Cook, R., Gilbert, K., Harrison, D., Hoang, L., Keagle,
P., Lumm, W., Pothier, B., Qui, D., Spadafora, R., Vicaire, R., Wang,
Y., Wierzbowski, J., Gibson, R., Jiwani, N., Caruso, A., Bush, D.,
Hershel, S., Patwell, D., Prabhakar, S., McDougall, S., Shimer, G.,
Goyal, A., Pietrokovski, S., Church, G. M., Daniels, C. J., Mao, J.-I.,
Rice, P., Nolling, J., Reeve, J. N., et al. (1997) J. Bacteriol. 179, 7135-7155).
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM 51334 and Department of Energy Grant DE-FG02-87ER13651 (to R. S. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF039105.
To whom correspondence should be addressed: University of Illinois
at Urbana-Champaign, Dept. of Microbiology, B103 Chemical and Life
Sciences Laboratory, 601 S. Goodwin Ave., Urbana, IL 61801. Tel.:
217-333-1397; Fax: 217-244-8485; E-mail:
biswarup_mukhopadhyay{at}qms1.life.uiuc.edu.
§ Present address: Archer Daniels Midland Co., Technical Center, 1001 Brush College Rd., Decatur, IL 62521.
1 The abbreviations used are: PYC, pyruvate carboxylase; PEP, phosphoenolpyruvate; PPC, PEP, carboxylase; PYCA, A subunit of PYC; PYCB, B subunit of PYC; ORF, open reading frame; OAD, oxaloacetate decarboxylase; TC, (S)-methylmalonyl-CoA-pyruvate transcarboxylase; PCC, propionyl-CoA carboxylase; ACC, acetyl-CoA carboxylase; BC, biotin carboxylase; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; MES, 4-morpholineethanesulfonic acid; bp, base pairs; kb, kilobase pairs.
2 A. A. DiMarco and J. E. Cronan, Jr., personal communication.
3 Sequence is available on-line at the following address: http//:WWW.genomecorp.com/htdocs/sequences/methanobacter/abstract.html
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|