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PubMed2001

Radical Shuttling in a Protein: Ribose Pseudorotation Controls Alkyl-Radical Transfer in the Coenzyme B(12) Dependent Enzyme Glutamate Mutase This work was supported by the Österreichische Akademie der Wissenschaften (APART fellowship 614), the Österreichische Fonds zur Förderung der wissenschaftlichen Forschung (FWF-project 11599), and the European Commission (TMR project number ERB 4061 PL 95-0307). Crystallographic data were collected at the EMBL-beamline BW7B at DESY in Hamburg, Germany. We thank the beamline scientists for their assistance, and Ingrid Dreveny, Günter Gartler, Gerwald Jogl, and Oliver Sauer for their help during data collection. This research emerged from a collaboration with Prof. W. Buckel (Marburg) who supplied us with clones of the glutamate mutase proteins.

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Journal Article↗
PubMed1987

Immunochemical studies of fibroblasts from patients with methylmalonyl-CoA mutase apoenzyme deficiency: detection of a mutation interfering with mitochondrial import.

Methylmalonyl-CoA mutase (2-methylmalonyl-CoA CoA-carbonylmutase, EC 5.4.99.2) is a mitochondrial enzyme whose deficiency in man leads to several biochemically and clinically heterogenous++ forms of methylmalonic acidemia. Intact fibroblasts from 21 patients with mutase apoenzyme deficiency have been pulse-labeled with [3H]leucine or [35S]methionine to determine how amounts of newly synthesized mutase recovered from these cells by immunoprecipitation compare with the amounts of steady-state crossreacting material previously determined. Ten lines (3 mut-, 7 mut 0 ), previously shown to have detectable steady-state crossreacting material, had amounts of newly synthesized mutase that varied from similar (7 lines) to considerably greater than (3 lines) the steady-state amounts. Of 11 lines that had no detectable steady-state crossreacting material, 6 had no detectable newly synthesized mutase, and 5 had amounts of mutase ranging from just detectable to almost half that of control. This result suggests that, at least for this latter group, one effect of the mutation in the mutase gene is to reduce the stability of the mutase protein. We examined fibroblasts from 48 patients with mutase apoenzyme deficiency to determine the sizes of the mature mutase subunit and the mutase precursor accumulated in the presence of the mitochondrial transport inhibitor rhodamine 6G. Of the 38 lines that had detectable newly synthesized mutase, only 2, lines 437 and 552, showed a pattern different from that generated by the normal precursor and mature subunits. Line 437 had two immunoprecipitable precursor proteins in the presence of rhodamine, each of which appeared to be transported and processed in the cells to produce two distinct mature proteins. Line 552 also had two anti-mutase reactive proteins in the presence of rhodamine, but each was smaller than the normal mature subunit and neither appeared to be proteolytically processed. The defect in line 552 is almost certainly an amino-terminal deletion that removes the leader peptide necessary for proper uptake and cleavage of the mutase precursor; this represents a clear example of a natural human mutation that interferes with mitochondrial transport of a protein.

Cell Line↗
PubMed2005

Purified recombinant hypothetical protein coded by open reading frame Rv1885c of Mycobacterium tuberculosis exhibits a monofunctional AroQ class of periplasmic chorismate mutase activity.

Naturally occurring variants of the enzyme chorismate mutase are known to exist that exhibit diversity in enzyme structure, regulatory properties, and association with other proteins. Chorismate mutase was not annotated in the initial genome sequence of Mycobacterium tuberculosis (Mtb) because of low sequence similarity between known chorismate mutases. Recombinant protein coded by open reading frame Rv1885c of Mtb exhibited chorismate mutase activity in vitro. Biochemical and biophysical characterization of the recombinant protein suggests its resemblance to the AroQ class of chorismate mutases, prototype examples of which include the Escherichia coli and yeast chorismate mutases. We also demonstrate that unlike the corresponding proteins of E. coli, Mtb chorismate mutase does not have any associated prephenate dehydratase or dehydrogenase activity, indicating its monofunctional nature. The Rv1885c-encoded chorismate mutase showed allosteric regulation by pathway-specific as well as cross-pathway-specific ligands, as evident from proteolytic cleavage protection and enzyme assays. The predicted N-terminal signal sequence of Mtb chorismate mutase was capable of functioning as one in E. coli, suggesting that Mtb chorismate mutase belongs to the AroQ class of chorismate mutases. It was evident that Rv1885c may not be the only enzyme with chorismate mutase enzyme function within Mtb, based on our observation of the presence of chorismate mutase activity displayed by another hypothetical protein coded by open reading frame Rv0948c, a novel instance of the existence of two monofunctional chorismate mutases ever reported in any pathogenic bacterium.

Allosteric Regulation↗
PubMed1989

The phosphoglycerate mutases.

The phosphoglycerate mutase family is generally very well documented with respect to structure, evolution, and mode of action. However, a few individuals in the family remain relatively poorly characterized and will clearly require more detailed study. Furthermore, certain aspects of the detailed behavior of these enzymes are, as yet, incompletely understood and require further investigation. Cofactor-dependent monophosphoglycerate mutase and bisphosphoglycerate mutase are undoubtedly very closely related. Their amino acid sequences are strongly similar, they can form active heterodimers, and they catalyze the same three reactions, albeit at substantially different relative rates. Both enzymes catalyze a ping-pong type of reaction with a phosphohistidine intermediate. The presence of an additional phospho ligand at the active site of monophosphoglycerate mutase helps to explain why this enzyme is better at retaining the 2,3-bisphosphoglycerate intermediate and why it is thus more efficient (by a factor of about 10(3)) at catalyzing the interconversion of 3- and 2-phosphoglycerates. The reason why 1,3-bisphosphoglycerate is a better substrate for bisphosphoglycerate mutase than for monophosphoglycerate mutase (by a factor of about 30) is not yet apparent but presumably relates to the relative positioning of the two phospho-binding sites. Both enzymes are equally good as phosphatases when the reaction is activated by 2-phosphoglycollate. Available evidence indicates that these mutases are similar in many respects to the much smaller, cofactor-dependent monophosphoglycerate mutase from Schizosaccharomyces pombe, but further information is required to define the relationship more precisely. Cofactor-independent monophosphoglycerate mutase belongs to a quite distinct branch of the phosphoglycerate mutase family. It is not known at present whether this branch is related divergently or convergently to the cofactor-dependent monophosphoglycerate mutase/bisphosphoglycerate mutase branch. Existing evidence can be argued both ways. For example, the kinetic evidence shows a ping-pong type of reaction and would be consistent with a phosphohistidine intermediate as encountered in the other mutases. Thus the cofactor-independent enzyme may also have arisen by gene duplication--but, in this case, yielding an enzyme of about twice the size, with slightly different residues at the active site and C-terminal tail. An alternative possibility, of course, is that the two branches of the phosphoglycerate mutase family are quite unrelated in a divergent sense and are little more similar structurally than is, for example, the catalytically similar enzyme phosphoglucomutase.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2004

MeaB is a component of the methylmalonyl-CoA mutase complex required for protection of the enzyme from inactivation.

Adenosylcobalamin-dependent methylmalonyl-CoA mutase catalyzes the interconversion of methylmalonyl-CoA and succinyl-CoA. In humans, deficiencies in the mutase lead to methylmalonic aciduria, a rare disease that is fatal in the first year of life. Such inherited deficiencies can result from mutations in the mutase structural gene or from mutations that impair the acquisition of cobalamins. Recently, a human gene of unknown function, MMAA, has been implicated in methylmalonic aciduria (Dobson, C. M., Wai, T., Leclerc, D., Wilson, A., Wu, X., Dore, C., Hudson, T., Rosenblatt, D. S., and Gravel, R. A. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 15554-15559). MMAA orthologs are widespread in bacteria, archaea, and eukaryotes. In Methylobacterium extorquens AM1, a mutant defective in the MMAA homolog meaB was unable to grow on C(1) and C(2) compounds because of the inability to convert methylmalonyl-CoA to succinyl-CoA (Korotkova N., Chistoserdova, L., Kuksa, V., and Lidstrom, M. E. (2002) J. Bacteriol. 184, 1750-1758). Here we demonstrate that this defect is not due to the absence of adenosylcobalamin but due to an inactive form of methylmalonyl-CoA mutase. The presence of active mutase in double mutants defective in MeaB and in the synthesis of either R-methylmalonyl-CoA or adenosylcobalamin indicates that MeaB is necessary for protection of mutase from inactivation during catalysis. MeaB and methylmalonyl-CoA mutase from M. extorquens were cloned and purified in their active forms. We demonstrated that MeaB forms a complex with methylmalonyl-CoA mutase and stimulates in vitro mutase activity. These results support the hypothesis that MeaB functions to protect methylmalonyl-CoA mutase from irreversible inactivation.

Burkholderia↗
PubMed1980

Inherited methylmalonyl CoA mutase apoenzyme deficiency in human fibroblasts: evidence for allelic heterogeneity, genetic compounds, and codominant expression.

We have measured and characterized methylmalonyl coenzyme A (CoA) mutase activity in extracts of cultured human fibroblasts from 23 patients with inherited deficiency of the mutase apoenzyme and from eight obligate heterozygotes for this defect. The mutant cell lines fall into two categories. Those without detectable residual mutase activity in cell extracts (>0.1% of control), and whose ability to utilize propionate in intact cells is refractory to supplementation of the culture medium with hydroxocobalamin, are designated mut degrees mutants. Those with detectable residual activity in cell extracts ( approximately 0.5-50% of control), and whose ability to utilize propionate in intact cells in markedly increased by hydroxocobalamin supplementation, are designated mut(-) mutants. The mutant enzyme in the mut(-) mutants exhibits a 50- to 5,000-fold elevated Michaelis constant (K(m)) for adenosylcobalamin in vitro, a normal K(m) for methylmalonyl CoA, and a strikingly reduced thermal stability at 45 degrees C relative to control. Mutase from one mut(-) mutant turns over at a rate three to four times that of control enzyme when cells are grown in hydroxocobalamin-supplemented medium.To detect heterozygous carriers of mutant mut alleles, we compared mutase activity in fibroblast extracts from four controls with that from eight parents of either mut degrees or mut(-) mutants. After cell growth in either unsupplemented or hydroxocobalamin-supplemented medium, activity in cell lines from heterozygotes was reduced to 47 or 37% of the mean control activities, respectively. We also examined the effect of adenosylcobalamin concentration on reaction kinetics of mutase from heterozygote cell lines. All four cell lines from parents of mut(-) mutants exhibited complex enzyme kinetics; approximately 80% of mutase activity demonstrated a K(m) indistinguishable from control, whereas a smaller component of activity exhibited a K(m) similar to the abnormal K(m) expressed by the mut(-) propositus in each family. In two families with a mut degrees propositus, mutase from three of the four parents exhibited only the normal K(m) for adenosylcobalamin, whereas mutase from one parent displayed complex kinetics, indicating expression of both a normal allele (mut(+)) and a mutant allele with an abnormal K(m). From these studies, we conclude that mut mutants reflect mutations at the autosomal gene locus for the methylmalonyl CoA mutase apoenzyme; that mut degrees , mut(-), and mut(+) alleles at this locus are codominantly expressed; and that some mut mutants may be genetic compounds, inheriting two different mut degrees or mut(-) alleles from their parents.

Alleles↗
PubMed1984

Biogenesis of the mitochondrial enzyme methylmalonyl-CoA mutase. Synthesis and processing of a precursor in a cell-free system and in cultured cells.

Methylmalonyl-CoA mutase (EC 5.4.99.2; mutase), a cytoplasmically synthesized mitochondrial matrix enzyme, is translated in a cell-free system programmed with rat liver RNA as a larger precursor polypeptide, designated pre-mutase, which appears to be 3-4 kDa larger than the subunit of purified mutase (77.5 kDa). When pre-mutase is incubated with intact rat liver mitochondria, it is taken up by them and proteolytically processed to the size of the mature subunit. The overall reaction is inhibited by compounds such as dinitrophenol which disrupt mitochondrial energy metabolism. The final, proteolytic step can be carried out by the mitochondrial matrix in the presence of added Zn2+ and is inhibited by metal ion chelators and by certain protease inhibitors (e.g. leupeptin and p-aminobenzamidine). Newly synthesized mutase was also detected in intact, cultured Buffalo rat liver cells labeled with [3H] leucine in the presence of dinitrophenol. When dinitrophenol is removed in a pulse-chase protocol, the accumulated pre-mutase is rapidly (t1/2 = 6-9 min) converted to mutase. On the other hand, when the chase is performed in the presence of the inhibitor, the labeled pre-mutase persists for greater than 5 h. This long term stability of pre-mutase contrasts sharply with the instability previously reported for unprocessed precursors of two other mitochondrial enzymes.

Animals↗
PubMed1986

On the mechanism of action of methylmalonyl-CoA mutase. Change of the steric course on isotope substitution.

When (methyl-2H3)methylmalonyl-CoA was reacted with partially purified methylmalonyl-CoA mutase, 1H-NMR revealed that about 24% of the migrating deuterium was lost after 88% conversion. When [methyl-3H]methylmalonyl-CoA was incubated with highly purified methylmalonyl-CoA mutase, tritium exchange with the medium depended on added methylmalonyl-CoA epimerase. With highly purified preparations of methylmalonyl-CoA mutase, effective tritium exchange from [5'-3H]adenosylcobalamin to water required the addition of methylmalonyl-CoA epimerase and of substrate (e.g. succinyl-CoA). By addition of [14C]succinyl-CoA to a partially purified preparation of methylmalonyl-CoA mutase, it was shown that the mutase binds one substrate molecule very tightly. Coupling the mutase reaction with the transcarboxylase reaction and using variously labelled succinyl-CoA as substrate, revealed that only (2R)- and not (2S)-methylmalonyl-CoA will be formed by the mutase with a kinetic isotope effect of 3.5 using (2H4)succinyl-CoA. When (1-13C) propionyl-CoA was reacted with a mixture of highly purified methylmalonyl-CoA carboxylase, epimerase and mutase, 13C-NMR signals were obtained for the thioester carbonyl of succinyl-CoA (relative intensity 100%) and of methylmalonyl-CoA (5%) as well as for the carboxyl of free succinic acid (27%) and of succinyl-CoA (less than 4.5%). Thus very little, if any, migration of the CoA from one carboxyl to the other appears to take place. (1,4-13C2)Succinic acid and (1,4-13C2)succinyl-CoA were synthesised and their 13C-NMR chemical shifts were exactly determined. Evidence is provided for a strict stereospecificity of the mutase toward the (2R)-epimer of methylmalonyl-CoA and for an incomplete stereospecificity toward the two diastereotopic 3-H atoms of succinyl-CoA. The latter, combined with a high intramolecular isotope discrimination, causes rapid washing-out of the migrating 2H and 3H to water and slow washing-in from the medium. Whenever migration of protium from the sterically less preferred 3-pro(S)- position of succinyl-CoA occurs and simultaneously a heavy isotope is maneuvered from the migratable 3-pro(R)- position into the labile alpha-position of methylmalonyl-CoA, the substitution by the COSCoA group takes place with inversion of configuration. When the sterically preferred 3-pro(R)-hydrogen atom migrates, the previously reported stereochemical retention occurs. A mechanistic and stereochemical scheme is discussed that fully accounts for all observations.

Acyl Coenzyme A↗
PubMed1992

Cloning, overexpression and mechanistic studies of carboxyphosphonoenolpyruvate mutase from Streptomyces hygroscopicus.

The enzyme carboxyphosphonoenolpyruvate mutase catalyses the formation of one of the two C-P bonds in bialaphos, a potent herbicide isolated from Streptomyces hygroscopicus. The gene encoding the enzyme has been cloned from a subgenomic library from S. hygroscopicus by colony hybridisation using an exact nucleotide probe. An open reading frame has been identified that encodes a protein of molecular mass 32700 Da, in good agreement with the subunit molecular mass of the carboxyphosphonoenolpyruvate mutase recently isolated from this source [Hidaka, T., Imai, S., Hara, O., Anzai, H., Murakami, T., Nagaoka, K. & Seto, H. (1990) J. Bacteriol. 172, 3066-3072]. The gene shares significant sequence similarity with that of phosphoenolpyruvate mutase, an enzyme that catalyses the related interconversion of phosphoenolpyruvate and phosphonopyruvate. When the carboxyphosphonoenolpyruvate-mutase gene was subcloned into the vector pET11a, the mutase was expressed as about 20% of the total soluble cellular protein in Escherichia coli. The mutase has been purified to homogeneity in three steps in 40% yield. With malate dehydrogenase/NADH, (hydroxyphosphinyl)pyruvate gives (hydroxyphosphinyl)lactate (kcat 164 s-1 and Km 680 microM) and this spectrophotometric assay for the product of the mutase reaction has been employed in the mechanistic studies. The kinetics for the mutase reaction have been evaluated for the substrate, carboxyphosphonoenolpyruvate, and for the putative reaction intermediate carboxyphosphinopyruvate, both of which have been prepared by chemical synthesis. Carboxyphosphonoenolpyruvate is converted to (hydroxyphosphinyl)pyruvate with a kcat of 0.020 s-1 and a Km of 270 microM, and carboxyphosphinopyruvate is converted to (hydroxyphosphinyl)pyruvate with a kcat of 7.6 x 10(-4) s-1 and a Km of 2.2 microM. Although the exogenously added intermediate is not kinetically competent, these results suggest that the mechanism for the mutase reaction involves an initial rearrangement to the intermediate carboxyphosphinopyruvate, followed by decarboxylation to yield the product (hydroxyphosphinyl)pyruvate.

Amino Acid Sequence↗
PubMed2004

Crystal structure of human bisphosphoglycerate mutase.

Bisphosphoglycerate mutase is a trifunctional enzyme of which the main function is to synthesize 2,3-bisphosphoglycerate, the allosteric effector of hemoglobin. The gene coding for bisphosphoglycerate mutase from the human cDNA library was cloned and expressed in Escherichia coli. The protein crystals were obtained and diffract to 2.5 A and produced the first crystal structure of bisphosphoglycerate mutase. The model was refined to a crystallographic R-factor of 0.200 and R(free) of 0.266 with excellent stereochemistry. The enzyme remains a dimer in the crystal. The overall structure of the enzyme resembles that of the cofactor-dependent phosphoglycerate mutase except the regions of 13-21, 98-117, 127-151, and the C-terminal tail. The conformational changes in the backbone and the side chains of some residues reveal the structural basis for the different activities between phosphoglycerate mutase and bisphosphoglycerate mutase. The bisphosphoglycerate mutase-specific residue Gly-14 may cause the most important conformational changes, which makes the side chain of Glu-13 orient toward the active site. The positions of Glu-13 and Phe-22 prevent 2,3-bisphosphoglycerate from binding in the way proposed previously. In addition, the side chain of Glu-13 would affect the Glu-89 protonation ability responsible for the low mutase activity. Other structural variations, which could be connected with functional differences, are also discussed.

Amino Acid Sequence↗
PubMed1993

Cloning, sequencing, and expression of the gene encoding methylmalonyl-coenzyme A mutase from Streptomyces cinnamonensis.

In streptomycetes, the conversion of succinyl-coenzyme A (CoA) into methylmalonyl-CoA, catalyzed by methylmalonyl-CoA mutase, most likely represents an important source of building blocks for polyketide antibiotic biosynthesis. In this work, the structural gene for methylmalonyl-CoA mutase from Streptomyces cinnamonensis was cloned by using a heterologous gene probe encoding the mutase from Propionibacterium shermanii. A 5,732-bp fragment was sequenced, within which four open reading frames were identified on one DNA strand. The two largest (mutA and mutB) overlap by 1 nucleotide and encode proteins of 616 and 733 residues showing high amino acid sequence similarities to each other and to methylmalonyl-CoA mutases from P. shermanii and mammalian sources. The transcriptional start of the mutA-mutB message, determined by S1 mapping, coincides with the first nucleotide of the translational start codon. Evidence that these two open reading frames encode a functional mutase in S. cinnamonensis was obtained by subcloning and expression in Streptomyces lividans TK64. The mutA and mutB gene products were detected in Western blots (immunoblots) with mutase-specific antibodies and by direct detection of mutase activity with a newly developed assay method. The methylmalonyl-CoA mutase was unable to catalyze the conversion of isobutyryl-CoA into n-butyryl-CoA, another closely related adenosylcobalamin-dependent rearrangement known to occur in S. cinnamonensis.

Amino Acid Sequence↗
PubMed1992

Cloning and sequencing of a cDNA encoding 2,3-bisphosphoglycerate-independent phosphoglycerate mutase from maize. Possible relationship to the alkaline phosphatase family.

The primary sequence of maize 2,3-bisphosphoglycerate-independent phosphoglycerate mutase was deduced from cDNAs isolated from maize cDNA libraries by screening with specific antibodies to the cofactor-independent enzyme and from a maize genomic clone. The genomic clone provided the 5'-nucleotide sequence encoding the N-terminal amino acids which could not be obtained from the cDNA. Confirmation that the nucleotide sequence was for the cofactor-independent phosphoglycerate mutase was obtained by sequencing the peptides generated from cyanogen bromide cleavage of the purified protein. This is the first report of the amino acid sequence of a 2,3-bisphosphoglycerate cofactor-independent phosphoglycerate mutase, which consists of 559 amino acids and is twice the molecular size of the mammalian cofactor-dependent enzyme subunit. Analysis of the cofactor-independent phosphoglycerate mutase amino acid sequence revealed no identity with the cofactor-dependent mutase types. Northern blot analysis confirmed this difference since the maize cofactor-independent phosphoglycerate mutase cDNA did not hybridize with mRNA of the cofactor-dependent mutase. The lack of amino acid identity between cofactor-dependent and -independent enzymes is consistent with their different catalytic mechanisms and suggests that both enzymes are unrelated evolutionarily and arose from two independent ancestral genes. However, a constellation of residues which are involved in metal ion binding in various alkaline phosphatases is conserved in the maize cofactor-independent phosphoglycerate mutase, which suggests that the enzyme is a member of the alkaline phosphatase family of enzymes.

Alkaline Phosphatase↗
PubMed1989

Human bisphosphoglycerate mutase. Expression in Escherichia coli and use of site-directed mutagenesis in the evaluation of the role of the carboxyl-terminal region in the enzymatic mechanism.

Bisphosphoglycerate mutase is an erythrocyte-specific enzyme whose main function is to synthesize 2,3-diphosphoglycerate, the allosteric effector of hemoglobin. In addition to its main 2,3-diphosphoglycerate synthase activity, the enzyme displays phosphatase and mutase activities both involving 2,3-diphosphoglycerate in their reaction. The three activities have been demonstrated to be catalysed at a unique active site. To study the structure of such an active site we have developed a recombinant system producing mutants of human bisphosphoglycerate mutase in Escherichia coli, by site-directed mutagenesis. For this purpose the human bisphosphoglycerate mutase cDNA that we had previously cloned has been used to construct a procaryotic high level expression vector bearing the "tac" promoter. Human bisphosphoglycerate mutase produced in E. coli, a species which does not normally synthesize this enzyme, represented 8% of the total soluble bacterial protein and displayed the three catalytic activities (synthase, mutase, and phosphatase) characteristic of the enzyme. Since it has been suggested that the carboxyl-terminal region may be implicated in the catalytic activity of the enzyme, three variants deleted in this part of the protein were produced. Our results indicate that a minimal deletion of 7 amino acid residues in the carboxyl-terminal portion of the human bisphosphoglycerate mutase completely abolished the three catalytic activities of the enzyme. In contrast, the effects of the deletion of the last two lysine residues were limited to a 38% reduction in the synthase activity. These results show that the carboxyl-terminal amino acid residues are either directly or indirectly implicated in the three catalytic functions of the human bisphosphoglycerate mutase, and that the two terminal lysine residues are not essential for the major part of the enzymatic mechanism of the enzyme.

Amino Acid Sequence↗