Essential Acidic Residues Research Articles

Proton-coupled transport mechanism of the efflux pump NorA.

Efflux pump antiporters confer drug resistance to bacteria by coupling proton import with the expulsion of antibiotics from the cytoplasm. Despite efforts there remains a lack of understanding as to how acid/base chemistry drives drug efflux. Here, we uncover the proton-coupling mechanism of the Staphylococcus aureus efflux pump NorA by elucidating structures in various protonation states of two essential acidic residues using cryo-EM. Protonation of Glu222 and Asp307 within the C-terminal domain stabilized the inward-occluded conformation by forming hydrogen bonds between the acidic residues and a single helix within the N-terminal domain responsible for occluding the substrate binding pocket. Remarkably, deprotonation of both Glu222 and Asp307 is needed to release interdomain tethering interactions, leading to opening of the pocket for antibiotic entry. Hence, the two acidic residues serve as a "belt and suspenders" protection mechanism to prevent simultaneous binding of protons and drug that enforce NorA coupling stoichiometry and confer antibiotic resistance.

Chemical Conformation of the Essential Glutamate Site of the c-Ring within Thermophilic Bacillus FoF1-ATP Synthase Determined by Solid-State NMR Based on its Isolated c-Ring Structure.

Proton translocation through the membrane-embedded Fo component of F-type ATP synthase (FoF1) is facilitated by the rotation of the Fo c-subunit ring (c-ring), carrying protons at essential acidic amino acid residues. Cryo-electron microscopy (Cryo-EM) structures of FoF1 suggest a unique proton translocation mechanism. To elucidate it based on the chemical conformation of the essential acidic residues of the c-ring in FoF1, we determined the structure of the isolated thermophilic Bacillus Fo (tFo) c-ring, consisting of 10 subunits, in membranes by solid-state NMR. This structure contains a distinct proton-locking conformation, wherein Asn23 (cN23) CγO and Glu56 (cE56) CδOH form a hydrogen bond in a closed form. We introduced stereo-array-isotope-labeled (SAIL) Glu and Asn into the tFoc-ring to clarify the chemical conformation of these residues in tFoF1-ATP synthase (tFoF1). Two well-separated 13C signals could be detected for cN23 and cE56 in a 505 kDa membrane protein complex, respectively, thereby suggesting the presence of two distinct chemical conformations. Based on the signal intensity and structure of the tFoc-ring and tFoF1, six pairs of cN23 and cE56 surrounded by membrane lipids take the closed form, whereas the other four in the a-c interface employ the deprotonated open form at a proportion of 87%. This indicates that the a-c interface is highly hydrophilic. The pKa values of the four cE56 residues in the a-c interface were estimated from the cN23 signal intensity in the open and closed forms and distribution of polar residues around each cE56. The results favor a rotation of the c-ring for ATP synthesis.

Residues in the Nucleosome Acidic Patch Regulate Histone Occupancy and Are Important for FACT Binding in Saccharomyces cerevisiae.

The essential histone chaperone FACT plays a critical role in DNA replication, repair, and transcription, primarily by binding to histone H2A-H2B dimers and regulating their assembly into nucleosomes. While FACT histone chaperone activity has been extensively studied, the exact nature of the H2A and H2B residues important for FACT binding remains controversial. In this study, we characterized the functions of residues in the histone H2A and H2B acidic patch, which is important for binding many chromatin-associated factors. We found that mutations in essential acidic patch residues cause a defect in histone occupancy in yeast, even though most of these histone mutants are expressed normally in yeast and form stable nucleosomes in vitro Instead, we show that two acidic patch residues, H2B L109 and H2A E57, are important for histone binding to FACT in vivo We systematically screened mutants in other H2A and H2B residues previously suspected to be important for FACT binding and confirmed the importance of H2B M62 using an in-vivo FACT-binding assay. Furthermore, we show that, like deletion mutants in FACT subunits, an H2A E57 and H2B M62 double mutant is lethal in yeast. In summary, we show that residues in the nucleosome acidic patch promote histone occupancy and are important for FACT binding to H2A-H2B dimers in yeast.

E. Coli F1Fo ATP Synthase Subunit C: Solution NMR Structure by CS-Rosetta

The ATP synthase is the main source of ATP in cells, whose function is to inter-convert chemical energy and transmembrane electrochemical potential difference. The enzyme contains two domains: a soluble F1 domain where ATP is synthesized/hydrolyzed and a transmembrane FO domain, which functions as an ion pump. Multiple (9-15) copies of subunit c form an oligomeric ring in the FO domain. The ion translocation activity takes place at the interface of c-ring and subunit a where an essential acidic residue in middle of the outer helix in subunit c undergoes a protonation/deprotonation process, which couples with the relative rotation of c-ring to subunit a. Although high-resolution X-ray structures of the c-rings from several organisms have been solved, the mechanism of ion translocation still needs to be clarified at atomic resolution.We aim to solve the structures of E. coli subunit c at both protonation states of the essential Asp61 and elucidate any conformational changes at the active site during the ion translocation process. Due to the difficulty of obtaining unambiguous long-range NOE constraints in membrane proteins, traditional solution NMR methods were unsuccessful in this case. We employed CS-Rosetta, which utilizes minimal NMR constraints, in our study.Our study of the subunit c in the protonated state shows that the choice of proper scoring weights is essential in the application of CS-Rosetta to membrane proteins. Using limited NMR restraints this method converges on a structure for E. coli subunit c that is similar to the X-ray structures of subunit c from other organisms. Further improving the quality of the structure may be achieved by optimizing the weights in the calculation. We are also using this approach on deprotonated subunit c and hoping to model the c-ring from our results.

Crystal structure of 1,3Gal43A, an exo-β-1,3-galactanase from Clostridium thermocellum

Glycoside hydrolase family 43 (GH43) consists of a variety of enzymes distributed widely in prokaryotes and eukaryotes. The mechanism by which GH43 enzymes hydrolyze oligosaccharides requires three essential acidic amino acid residues. However, one of them is thought to be missing in galactan β-1,3-galactosidases from the GH43 family. Ct1,3Gal43A, from Clostridium thermocellum, is comprised of a GH43 domain, a CBM13 domain, and a dockerin domain and exhibits an unusual ability to hydrolyze β-1,3-galactan in the presence of a β-1,6 linked branch. Here, we present its crystal structure at 2.7 Å resolution and complex structures of the enzyme with several substrates and analogs. Two modes of substrate binding were observed at the β site of the CtCBM13 domain, and one galactobiose molecule was found in an "L" shaped pocket of the CtGH43 domain, which appears large enough to accommodate two more galactose units. In addition, we found that mutating Glu112 to Gln or Ala eliminated the galactan hydrolysis activity of Ct1,3Gal43A while did not disrupt its ligand binding ability. Combining this results and the crystal structure we identified Glu112 in Ct1,3Gal43A as the 'missing' essential acidic residue in galactan β-1,3-galactosidases. Structural information presented here also suggests a mechanism by which Ct1,3Gal43A bypasses β-1,6 linked branches in the substrate and another mechanism by which the substrate is delivered 'in trans' from the CBM13 domain to the catalytic GH43 domain.

Genetic Characterization of Conserved Charged Residues in the Bacterial Flagellar Type III Export Protein FlhA

For assembly of the bacterial flagellum, most of flagellar proteins are transported to the distal end of the flagellum by the flagellar type III protein export apparatus powered by proton motive force (PMF) across the cytoplasmic membrane. FlhA is an integral membrane protein of the export apparatus and is involved in an early stage of the export process along with three soluble proteins, FliH, FliI, and FliJ, but the energy coupling mechanism remains unknown. Here, we carried out site-directed mutagenesis of eight, highly conserved charged residues in putative juxta- and trans-membrane helices of FlhA. Only Asp-208 was an essential acidic residue. Most of the FlhA substitutions were tolerated, but resulted in loss-of-function in the ΔfliH-fliI mutant background, even with the second-site flhB(P28T) mutation that increases the probability of flagellar protein export in the absence of FliH and FliI. The addition of FliH and FliI allowed the D45A, R85A, R94K and R270A mutant proteins to work even in the presence of the flhB(P28T) mutation. Suppressor analysis of a flhA(K203W) mutation showed an interaction between FlhA and FliR. Taken all together, we suggest that Asp-208 is directly involved in PMF-driven protein export and that the cooperative interactions of FlhA with FlhB, FliH, FliI, and FliR drive the translocation of export substrate.

A Bicarbonate Cofactor Modulates 1,4-Dihydroxy-2-naphthoyl-Coenzyme A Synthase in Menaquinone Biosynthesis of Escherichia coli

1,4-Dihydroxy-2-naphthoyl coenzyme A (DHNA-CoA) synthase is a typical crotonase-fold protein catalyzing an intramolecular Claisen condensation in the menaquinone biosynthetic pathway. We have characterized this enzyme from Escherichia coli and found that it is activated by bicarbonate in a concentration-dependent manner. The bicarbonate binding site has been identified in the crystal structure of a virtually identical ortholog (96.8% sequence identity) from Salmonella typhimurium through comparison with a bicarbonate-insensitive orthologue. Kinetic properties of the enzyme and its site-directed mutants of the bicarbonate binding site indicate that the exogenous bicarbonate anion is essential to the enzyme activity. With this essential catalytic role, the simple bicarbonate anion is an enzyme cofactor, which is usually a small organic molecule derived from vitamins, a metal ion, or a metal-containing polyatomic anionic complex. This finding leads to classification of the DHNA-CoA synthases into two evolutionarily conserved subfamilies: type I enzymes that are bicarbonate-dependent and contain a conserved glycine at the bicarbonate binding site; and type II enzymes that are bicarbonate-independent and contain a conserved aspartate at the position similar to the enzyme-bound bicarbonate. In addition, the unique location of the enzyme-bound bicarbonate allows it to be proposed as a catalytic base responsible for abstraction of the α-proton of the thioester substrate in the enzymatic reaction, suggesting a unified catalytic mechanism for all DHNA-CoA synthases.

The MrpA, MrpB and MrpD subunits of the Mrp antiporter complex in Bacillus subtilis contain membrane-embedded and essential acidic residues

Bacillus subtilis Mrp is a unique Na+/H+ antiporter with a multicomponent structure consisting of the mrpABCDEFG gene products. We have previously reported that the conserved and putative membrane-embedded Glu-113, Glu-657, Asp-743 and Glu-747 of MrpA (ShaA) are essential for the transport function. In this study, we further investigated the functional involvement of the equivalent conserved acidic residues of other Mrp proteins in heterologous Escherichia coli and natural B. subtilis backgrounds. Asp-121 of MrpB and Glu-137 of MrpD were additionally identified to be essential for the transport function in both systems. Glu-137 of MrpD and Glu-113 of MrpA were found to be conserved in the homologous MrpD/MrpA proteins as well as in the homologous subunits of H+-translocating primary active transporters such as Nuo and Mbh, suggesting their critical role in ion binding. The remaining essential acidic residues clustered in the C-terminal domain of MrpA (Glu-657, Asp-743 and Glu-747) and MrpB (Asp-121); these subunits are fused in some Gram-negative species. It is possible that the MrpA, MrpB and MrpD subunits, which contain essential transmembrane acidic residues, form the ion translocation site(s) of the Mrp antiporter complex.

Electroneutral and electrogenic catalysis by dihaem-containing succinate:quinone oxidoreductases

Membrane protein complexes can support both the generation and utilization of a transmembrane electrochemical proton potential (Deltap), either by supporting transmembrane electron transfer coupled to protolytic reactions on opposite sides of the membrane or by supporting transmembrane proton transfer. Regarding the first mechanism, this has been unequivocally demonstrated to be operational for Deltap-dependent catalysis of succinate oxidation by quinone in the case of the dihaem-containing SQR (succinate:menaquinone reductase) from the Gram-positive bacterium Bacillus licheniformis. This is physiologically relevant in that it allows the transmembrane Deltap to drive the endergonic oxidation of succinate by menaquinone by the dihaem-containing SQR of Gram-positive bacteria. In the case of a related but different respiratory membrane protein complex, the dihaem-containing QFR (quinol:fumarate reductase) of the epsilon-proteobacterium Wolinella succinogenes, evidence has been obtained indicating that both mechanisms are combined, so as to facilitate transmembrane electron transfer by proton transfer via a both novel and essential compensatory transmembrane proton transfer pathway ('E-pathway'). This is necessary because, although the reduction of fumarate by menaquinol is exergonic, it is obviously not exergonic enough to support the generation of a Deltap. This compensatory E-pathway appears to be required by all dihaem-containing QFR enzymes and the conservation of the essential acidic residue on transmembrane helix V (Glu-C180 in W. succinogenes QFR) is a useful key for the sequence-based discrimination of these QFR enzymes from the dihaem-containing SQR enzymes.

No Single Irreplaceable Acidic Residues in the Escherichia coli Secondary Multidrug Transporter MdfA

The largest family of solute transporters (major facilitator superfamily [MFS]) includes proton-motive-force-driven secondary transporters. Several characterized MFS transporters utilize essential acidic residues that play a critical role in the energy-coupling mechanism during transport. Surprisingly, we show here that no single acidic residue plays an irreplaceable role in the Escherichia coli secondary multidrug transporter MdfA.

EDEM3, a Soluble EDEM Homolog, Enhances Glycoprotein Endoplasmic Reticulum-associated Degradation and Mannose Trimming

Quality control in the endoplasmic reticulum ensures that only properly folded proteins are retained in the cell through mechanisms that recognize and discard misfolded or unassembled proteins in a process called endoplasmic reticulum-associated degradation (ERAD). We previously cloned EDEM (ER degradation-enhancing alpha-mannosidase-like protein) and showed that it accelerates ERAD of misfolded glycoproteins. We now cloned mouse EDEM3, a soluble homolog of EDEM. EDEM3 consists of 931 amino acids and has all the signature motifs of Class I alpha-mannosidases (glycosyl hydrolase family 47) in its N-terminal domain and a protease-associated motif in its C-terminal region. EDEM3 accelerates glycoprotein ERAD in transfected HEK293 cells, as shown by increased degradation of misfolded alpha1-antitrypsin variant (null (Hong Kong)) and of TCRalpha. Overexpression of EDEM3 also greatly stimulates mannose trimming not only from misfolded alpha1-AT null (Hong Kong) but also from total glycoproteins, in contrast to EDEM, which has no apparent alpha1,2-mannosidase activity. Furthermore, overexpression of the E147Q EDEM3 mutant, which has the mutation in one of the conserved acidic residues essential for enzyme activity of alpha1,2-mannosidases, abolishes the stimulation of mannose trimming and greatly decreases the stimulation of ERAD by EDEM3. These results show that EDEM3 has alpha1,2-mannosidase activity in vivo, suggesting that the mechanism whereby EDEM3 accelerates glycoprotein ERAD is different from that of EDEM.

Rotation of the c Subunit Oligomer in EF0EF1 Mutant cD61N

ATP synthases (F(0)F(1)-ATPases) mechanically couple ion flow through the membrane-intrinsic portion, F(0), to ATP synthesis within the peripheral portion, F(1). The coupling most probably occurs through the rotation of a central rotor (subunits c(10)epsilon gamma) relative to the stator (subunits ab(2)delta(alpha beta)(3)). The translocation of protons is conceived to involve the rotation of the ring of c subunits (the c oligomer) containing the essential acidic residue cD61 against subunits ab(2). In line with this notion, the mutants cD61N and cD61G have been previously reported to lack proton translocation. However, it has been surprising that the membrane-bound mutated holoenzyme hydrolyzed ATP but without translocating protons. Using detergent-solubilized and immobilized EF(0)F(1) and by application of the microvideographic assay for rotation, we found that the c oligomer, which carried a fluorescent actin filament, rotates in the presence of ATP in the mutant cD61N just as in the wild type enzyme. This observation excluded slippage among subunit gamma, the central rotary shaft, and the c oligomer and suggested free rotation without proton pumping between the oligomer and subunit a in the membrane-bound enzyme.

Identification of essential acidic residues of outer membrane protease OmpT supports a novel active site

Escherichia coli outer membrane protease OmpT has previously been classified as a serine protease with Ser99 and His212 as active site residues. The recently solved X-ray structure of the enzyme was inconsistent with this classification, and the involvement of a nucleophilic water molecule was proposed. Here, we substituted all conserved aspartate and glutamate residues by alanines and measured the residual enzymatic activities of the variants. Our results support the involvement of a nucleophilic water molecule that is activated by the Asp210/His212 catalytic dyad. Activity is also strongly dependent on Asp83 and Asp85. Both may function in binding of the water molecule and/or oxyanion stabilization. The proposed mechanism implies a novel proteolytic catalytic site.

Involvement of phylogenetically conserved acidic amino acid residues in catalysis by an oxidative DNA damage enzyme formamidopyrimidine glycosylase.

Formamidopyrimidine glycosylase (Fpg) is an important bacterial base excision repair enzyme, which initiates removal of damaged purines such as the highly mutagenic 8-oxoguanine. Similar to other glycosylase/AP lyases, catalysis by Fpg is known to proceed by a nucleophilic attack by an amino group (the secondary amine of its N-terminal proline) on C1' of the deoxyribose sugar at a damaged base, which results in the departure of the base from the DNA and removal of the sugar ring by beta/delta-elimination. However, in contrast to other enzymes in this class, in which acidic amino acids have been shown to be essential for glycosyl and phosphodiester bond scission, the catalytically essential acidic residues have not been documented for Fpg. Multiple sequence alignments of conserved acidic residues in all known bacterial Fpg-like proteins revealed six conserved glutamic and aspartic acid residues. Site-directed mutagenesis was used to change glutamic and aspartic acid residues to glutamines and asparagines, respectively. While the Asp to Asn mutants had no effect on the incision activity on 8-oxoguanine-containing DNA, several of the substitutions at glutamates reduced Fpg activity on the 8-oxoguanosine DNA, with the E3Q and E174Q mutants being essentially devoid of activity. The AP lyase activity of all of the glutamic acid mutants was slightly reduced as compared to the wild-type enzyme. Sodium borohydride trapping of wild-type Fpg and its E3Q and E174Q mutants on 8-oxoguanosine or AP site containing DNA correlated with the relative activity of the mutants on either of these substrates.

Crystal structure of a class I alpha1,2-mannosidase involved in N-glycan processing and endoplasmic reticulum quality control.

Mannose trimming is not only essential for N-glycan maturation in mammalian cells but also triggers degradation of misfolded glycoproteins. The crystal structure of the class I alpha1, 2-mannosidase that trims Man(9)GlcNAc(2) to Man(8)GlcNAc(2 )isomer B in the endoplasmic reticulum of Saccharomyces cerevisiae reveals a novel (alphaalpha)(7)-barrel in which an N-glycan from one molecule extends into the barrel of an adjacent molecule, interacting with the essential acidic residues and calcium ion. The observed protein-carbohydrate interactions provide the first insight into the catalytic mechanism and specificity of this eukaryotic enzyme family and may be used to design inhibitors that prevent degradation of misfolded glycoproteins in genetic diseases.

Roles of acidic residues in the hydrophilic loop regions of metal-tetracycline/H + antiporter Tet(K) of Staphylococcus aureus

Three transmembrane glutamic acid residues play essential roles in the metal-tetracycline/H + antiporter Tet(K) of Staphylococcus aureus [Fujihira et al., FEBS Lett. 391 (1996) 243–246]. In the putative hydrophilic loop region of the Tet(K) and Tet(L) proteins, six acidic residues are conserved. Asp 74, Asp 200, Asp 318 and Glu 381 are located on the putative cytoplasmic side, and Asp 39 and Glu 345 on the putative periplasmic side. These residues were replaced by a neutral amino acid residue or a charge-conserved one. In contrast to the transmembrane glutamic acid residues, the replacement of the two glutamic acid residues (Glu 345 and Glu 381) did not affect the tetracycline resistance level. Out of the other four aspartic acid residues, the only essential residue is Asp 318, any replacement of which resulted in complete loss of the tetracycline resistance and transport activity. Asp 318 is located in cytoplasmic loop 10–11 in the putative 14-transmembrane-segment topology of Tet(K). In the case of the tetracycline exporters of Gram-negative bacteria, the only essential acidic residue in the cytoplasmic loop region is located in loop 2–3 [Yamaguchi et al., Biochemistry 31 (1992) 8344–8348]. It may be a general role for tetracycline efflux proteins that three transmembrane and one cytoplasmic acidic residues are mandatory for the tetracycline transport function.

The type III connecting segment of fibronectin contains an aspartic acid residue that regulates the rate of binding to integrin alpha 4 beta 1.

The type III connecting segment (IIICS) within fibronectin is the major binding site for the integrin alpha 4 beta 1. Most integrin ligands have an essential acidic residue within their integrin binding site, in IIICS this residue is hypothesized to be the aspartic acid at position 21. Alanine scanning mutagenesis was used to determine the amino acid residues within the intact IIICS domain required for interaction with alpha 4 beta 1. IIICS was cloned and expressed as a fusion protein with glutathione S-transferase. This recombinant form of IIICS supports the adhesion of CHO cells that express human alpha 4 beta 1 in a cation dependent manner. Alanine scanning mutagenesis of the EILDVP sequence in recombinant IIICS demonstrated that only two of these residues are critical for adhesion of alpha 4 beta 1 expressing cells. Mutations of leucine at position 20 and aspartic acid at position 21 to alanine significantly reduced cell adhesion. Conservative mutations of aspartic acid at position 21 to asparagine or glutamic acid also reduced the ability of the recombinant protein to support cell adhesion, although not to the same extent as the corresponding alanine replacement. Most importantly, we show that although the mutation of asp 21 impairs cell adhesion, an examination of cell adhesion as a function of time demonstrated that asp 21 is not necessary for cell adhesion through alpha 4 beta 1. In comparison to wild type IIICS, the asp 21 to ala mutant supported minimal adhesion at early time points (10-30 min.), but was equivalent to wild type IIICS in supporting adhesion over one hour.

Identification of an essential acidic residue in Cdc25 protein phosphatase and a general three-dimensional model for a core region in protein phosphatases.

The reaction mechanism of protein tyrosine phosphatases (PTPases) and dual-specificity protein phosphatases is thought to involve a catalytic aspartic acid residue. This residue was recently identified by site-directed mutagenesis in Yersinia PTPase, VHR protein phosphatase, and bovine low molecular weight protein phosphatase. Herein we identify aspartic acid 383 as a potential candidate for the catalytic acid in human Cdc25A protein phosphatase, using sequence alignment, structural information, and site-directed mutagenesis. The D383N mutant enzyme exhibits a 150-fold reduction in kcat, with Kw only slightly changed. Analysis of sequence homologies between several members of the Cdc25 family and deletion mutagenesis substantiate the concept of a two-domain structure for Cdc25, with a regulatory N-terminal and a catalytic C-terminal domain. Based on the alignment of catalytic residues and secondary structure elements, we present a three-dimensional model for the core region of Cdc25. By comparing this three-dimensional model to the crystal structures of PTP1b, Yersinia PTPase, and bovine low molecular weight PTPase, which share only very limited amino acid sequence similarities, we identify a general architecture of the protein phosphatase core region, encompassing the active site loop motif HCXXXXXR and the catalytic aspartic acid residue.

Second-site suppressor mutations for the Asp-66-->Cys mutant of the transposon Tn10-encoded metal-tetracycline/H+ antiporter of Escherichia coli.

Asp66 is the only essential acidic residue in the putative hydrophilic loop2-3 region of the transposon Tn10-encoded metal-tetracycline/H+ antiporter [TetA(B)] [Yamaguchi, A., Nakatani, M., & Sawai, T. (1992a) Biochemistry 31, 8344-8348]. Escherichia coli cells producing a D66C mutant of TetA(B) showed no tetracycline resistance. A spontaneous second-site revertant was isolated from the cells carrying the D66C mutant gene, which showed moderate resistance to tetracycline [minimum inhibitory concentration (MIC), 50 micrograms/mL]. The entire sequencing of the revertant genes revealed two secondary mutations, i.e., the codon 40 of GCT (Ala)-->GAT (Asp) and T-->G at 17 bases upstream from the initiation codon of the tetA gene. There was a T-->G mutation at position -17, which was a mutation in the tet promoter/operator region, which caused a decrease in TetA(B) production. The full expression of the A40D/D66C double and the A40D single mutants, which were constructed by site-directed mutagenesis, was deleterious for cell growth. The -17T-->G mutation mitigated the deleterious effect of these mutants through reduction of expression. The -17T-->G single mutation introduced into the wild-type tet gene did not affect the level of resistance, although the expression was significantly reduced. Intact cells carrying the A40D/D66C and -17T-->G/A40D/D66C mutant plasmids showed a reduced level of tetracycline accumulation due to active efflux, whereas no significant tetracycline uptake was observed in inverted membrane vesicles prepared from these mutant-producing cells. The A40D mutation is located at opposite side of the membrane to the D66C mutation in the putative secondary structure of TetA(B). The second-site mutation might mediate its effects through a structural perturbation propagated along the polypeptide backbone.

The active site of hamster 3-hydroxy-3-methylglutaryl-CoA reductase resides at the subunit interface and incorporates catalytically essential acidic residues from separate polypeptides.

We employed site-directed mutagenesis based on sequence comparisons and characterization of purified mutant enzymes to identify Glu558 and Asp766 of Syrian hamster 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) as essential for catalysis. Mutant enzymes E558D, E558Q, and D766N had wild-type Km values for (S)-HMG-CoA and NADPH, but exhibited less than 0.5% of the wild-type catalytic activity. The inactive mutant polypeptides E558Q and D766N nevertheless can associate to generate an active enzyme. In vitro, 6% of the wild-type activity was observed when mutant polypeptides E558D and D766N were mixed in the absence of chaotropic agents. When mutant polypeptides E558Q and D766N were co-expressed in Escherichia coli, the resulting purified enzyme had 25% of wild-type activity. Hamster HMG-CoA reductase thus is a two-site, dimeric enzyme whose subunits associate to form an active site in which each monomer contributes at least one residue (e.g. Glu558 from one monomer and Asp766 from the other). The wild-type enzyme behaves as a dimer during size exclusion chromatography and has one HMG-CoA binding site per monomer. Syrian hamster HMG-CoA reductase thus appears to be a homodimer with two active sites which are located at the subunit interface.