Three dimensional structure of porcine pancreatic alpha-amylase at 2.9 A resolution. Role of calcium in structure and activity.

The crystal structure of porcine pancreatic alpha-amylase (PPA) has been solved at 2.9 A resolution by X-ray crystallographic methods. The enzyme contains three domains. The larger, in the N-terminal part, consists of 330 amino acid residues. This central domain has the typical parallel-stranded alpha-beta barrel structure (alpha beta)8, already found in a number of other enzymes like triose phosphate isomerase and pyruvate kinase. The C-terminal domain forms a distinct globular unit where the chain folds into an eight-stranded antiparallel beta-barrel. The third domain lies between a beta-strand and a alpha-helix of the central domain, in a position similar to those found for domain B in triose phosphate isomerase and pyruvate kinase. It is essentially composed of antiparallel beta-sheets. The active site is located in a cleft within the N-terminal central domain, at the carboxy-end of the beta-strands of the (alpha beta)8 barrel. Binding of various substrate analogues to the enzyme suggests that the amino acid residues involved in the catalytic reaction are a pair of aspartic acids. A number of other residues surround the substrate and seem to participate in its binding via hydrogen bonds and hydrophobic interactions. The 'essential' calcium ion has been located near the active site region and between two domains, each of them providing two calcium ligands. On the basis of sequence comparisons this calcium binding site is suggested to be a common structural feature of all alpha-amylases. It represents a new type of calcium-protein interaction pattern.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glucose-6-phosphate isomerase

Glucose-6-phosphate isomerase (EC 5.3.1.9) is a dimeric enzyme of molecular mass 132000 which catalyses the interconversion of D-glucose-6-phosphate and D-fructose-6-phosphate. The crystal structure of the enzyme from pig muscle has been determined at a nominal resolution of 2.6 A. The structure is of the alpha/beta type. Each subunit consists of two domains and the active site is in both the domain interface and the subunit interface (P.J. Shaw & H. Muirhead (1976), FEBS Lett. 65, 50-55). Each subunit contains 13 methionine residues so that cyanogen bromide cleavage will produce 14 fragments, most of which have been identified and at least partly purified. Sequence information is given for about one-third of the molecule from 5 cyanogen bromide fragments. One of the sequences includes a modified lysine residue. Modification of this residue leads to a parallel loss of enzymatic activity. A tentative fit of two of the peptides to the electron density map has been made. It seems possible that glucose-6-phosphate isomerase, triose phosphate isomerase and pyruvate kinase all contain a histidine and a glutamate residue at the active site.     

Three-dimensional structure of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum at 2.9 A resolution

The three-dimensional structure of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Rhodospirillum rubrum has been determined at 2.9 Å resolution by X-ray crystallographic methods. The MIR-electron density map was substantially improved by two-fold non-crystallographic symmetry averaging. The polypeptide chains in the dimer were traced using a graphics display system with the help of the BONES option in FRODO. The dimer has approximate dimensions of 50 x 72 x 105 Å. The enzyme subunit is a typical two-domain protein. The smaller, N-terminal domain consists of 137 amino acid residues and forms a central, mixed five-stranded β-sheet with α-helices on both sides of the sheet. The larger C-terminal domain consists of 329 amino acid residues. This domain has an eight-stranded parallel α/β barrel structure as found in triosephosphate isomerase and a number of other functionally non-related proteins. The active site in Rubisco determined by difference Fourier techniques and fitting of active site residues to the electron density map, is located at the carboxy-end of the β-strands in the α/β barrel of the C-terminal domain. There are few domain–domain interactions within the subunit. The interactions at the interface between the two subunits of the dimer are tight and extensive. There are tight contacts between the two C-terminal domains, which build up the core of the molecule. There are also interactions between the N-terminal domain of one subunit and the C-terminal domain of the second subunit, close to the active site.     

Characterization of a gene encoding a Pichia pastoris protein disulfide isomerase

Protein disulphide isomerases belong to the thioredoxin superfamily of protein-thiol oxidoreductases that have two double-cysteine redox-active sites and take part in protein folding in the endoplasmic reticulum (ER). We report here the cloning of a Pichia pastoris genomic DNA fragment (2919 bp) that encodes the full length of a protein disulphide isomerase (PpPDI). The deduced amino acid sequence of PDI consists of 517 residues and carries the two characteristic PDI-type redox-active domains -CGHC-, separated by 338 residues, and two potential N-glycosylation sites. The N-terminal end forms a putative signal sequence, and an acidic C-terminal region represents a possible calcium-binding domain. Together with the -HDEL ER retrieval sequence at the C-terminus, these features indicate that the gene encodes a redox-active ER-resident protein disulphide isomerase. The nucleotide sequence, which also contains two other open reading frames, has been submitted to the EMBL Nucleotide Sequence Database, Accession No. AJ302014.     

The 3.0 A crystal structure of xylose isomerase from Streptomyces olivochromogenes

The crystal structure of xylose isomerase [E.C. 5.3.1.5] from Streptomyces olivochromogenes has been determined to 3.0 A resolution. The crystals belong to space group P22(1)2(1) with unit cell parameters a = 98.7, b = 93.9, c = 87.7. The asymmetric unit contains half of a tetrameric molecule of 222 symmetry. The two-fold axis relating the two molecules in the asymmetric unit is close to where a crystallographic two-fold would be if the space group were I222. This causes the diffraction pattern to have strong I222 pseudo-symmetry, so all data were collected in this pseudo-space group. Since the sequence of this enzyme has not been reported, a polyalanine backbone has been fitted to the electron density. Xylose isomerase has two domains: the N-terminal domain is an eight-stranded alpha/beta barrel of 299 residues. The C-terminal domain is a large loop of 50 residues which is involved in intermolecular contacts. Comparison of xylose isomerase with the archetypical alpha/beta barrel protein, triose phosphate isomerase, reveals that the proteins overlap best when the third (alpha beta) strand of xylose isomerase is superimposed on the first (alpha beta) strand of triose phosphate isomerase. This same overlap has also been found between the muconate lactonising enzyme and triose phosphate isomerase [Goldman et al. (1987) J. Mol. Biol., in press].     

The crystal and solution studies of glucosamine-6-phosphate synthase from Candida albicans

Glucosamine 6-phosphate (GlcN-6-P) synthase is an ubiquitous enzyme that catalyses the first committed step in the reaction pathway that leads to formation of uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc), a precursor of macromolecules that contain amino sugars. Despite sequence similarities, the enzyme in eukaryotes is tetrameric, whereas in prokaryotes it is a dimer. The activity of eukaryotic GlcN-6-P synthase (known as Gfa1p) is regulated by feedback inhibition by UDP-GlcNAc, the end product of the reaction pathway, whereas in prokaryotes the GlcN-6-P synthase (known as GlmS) is not regulated at the post-translational level. In bacteria and fungi the enzyme is essential for cell wall synthesis. In human the enzyme is a mediator of insulin resistance. For these reasons, Gfa1p is a target in anti-fungal chemotherapy and in therapeutics for type-2 diabetes. The crystal structure of the Gfa1p isomerase domain from Candida albicans has been analysed in complex with the allosteric inhibitor UDP-GlcNAc and in the presence of glucose 6-phosphate, fructose 6-phosphate and an analogue of the reaction intermediate, 2-amino-2-deoxy-d-mannitol 6-phosphate (ADMP). A solution structure of the native Gfa1p has been deduced using small-angle X-ray scattering (SAXS). The tetrameric Gfa1p can be described as a dimer of dimers, with each half similar to the related enzyme from Escherichia coli. The core of the protein consists of the isomerase domains. UDP-GlcNAc binds, together with a metal cation, in a well-defined pocket on the surface of the isomerase domain. The residues responsible for tetramerisation and for binding UDP-GlcNAc are conserved only among eukaryotic sequences. Comparison with the previously studied GlmS from E. coli reveals differences as well as similarities in the isomerase active site. This study of Gfa1p focuses on the features that distinguish it from the prokaryotic homologue in terms of quaternary structure, control of the enzymatic activity and details of the isomerase active site.     

Three-dimensional structure of the bifunctional enzyme phosphoribosylanthranilate isomerase: indoleglycerolphosphate synthase from Escherichia coli refined at 2.0 A resolution.

The three-dimensional structure of the monomeric bifunctional enzyme N-(5'-phosphoribosyl)anthranilate isomerase:indole-3-glycerol-phosphate synthase from Escherichia coli has been refined at 2.0 A resolution, using oscillation film data obtained from synchrotron radiation. The model includes the complete protein (452 residues), two phosphate ions and 628 water molecules. The final R-factor is 17.3% for all observed data between 15 and 2 A resolution. The root-mean-square deviations from ideal bond lengths and bond angles are 0.010 A and 3.2 degrees, respectively. The structure of N-(5'-phosphoribosyl)anthranilate isomerase: indole-3-glycerol-phosphate synthase from E. coli comprises two beta/alpha-barrel domains that superimpose with a root-mean-square deviation of 2.03 A for 138 C alpha-pairs. The C-terminal domain (residues 256 to 452) catalyses the PRAI reaction and the N-terminal domain (residues 1 to 255) catalyses the IGPS reaction, two sequential steps in tryptophan biosynthesis. The enzyme has the overall shape of a dumb-bell, resulting in a surface area that is considerably larger than normally observed for monomeric proteins of this size. The active sites of the PRAI and the IGPS domains, both located at the C-terminal side of the central beta-barrel, contain equivalent binding sites for the phosphate moieties of the substrates N-(5'-phosphoribosyl) anthranilate and 1-(o-carboxyphenylamino)-1-deoxyribulose-5-phosphate. These two phosphate binding sites are identical with respect to their positions within the tertiary structure of the beta/alpha-barrel, the conformation of the residues involved in phosphate binding and the hydrogen-bonding network between the phosphate ions and the protein. The active site cavities of both domains contain similar hydrophobic pockets that presumably bind the anthranilic acid moieties of the substrates. These similarities of the tertiary structures and the active sites of the two domains provide evidence that N-(5'-phosphoribosyl)anthranilate isomerase:indole-3-glycerol-phosphate synthase from E. coli results from a gene duplication event of a monomeric beta/alpha-barrel ancestor.     

FKBP immunophilins and Alzheimer’s disease: A chaperoned affair

The FK506-binding protein (FKBP) family of immunophilins consists of proteins with a variety of protein–protein interaction domains and versatile cellular functions. Analysis of the functions of immunophilins has been the focus of studies in recent years and has led to the identification of various molecular pathways in which FKBPs play an active role. All FKBPs contain a domain with prolyl cis/trans isomerase (PPIase) activity. Binding of the immunosuppressant molecule FK506 to this domain inhibits their PPIase activity while mediating immune suppression through inhibition of calcineurin. The larger members, FKBP51 and FKBP52, interact with Hsp90 and exhibit chaperone activity that is shown to regulate steroid hormone signalling. From these studies it is clear that FKBP proteins are expressed ubiquitously but show relatively high levels of expression in the nervous system. Consistent with this expression, FKBPs have been implicated with both neuroprotection and neurodegeneration. This review will focus on recent studies involving FKBP immunophilins in Alzheimer’s-disease-related pathways.     

Sulfhydryl oxidation, not disulfide isomerization, is the principal function of protein disulfide isomerase in yeast Saccharomyces cerevisiae

Protein disulfide isomerase (PDI) is an essential protein folding assistant of the eukaryotic endoplasmic reticulum that catalyzes both the formation of disulfides during protein folding (oxidase activity) and the isomerization of disulfides that may form incorrectly (isomerase activity). Catalysis of thiol-disulfide exchange by PDI is required for cell viability in Saccharomyces cerevisiae, but there has been some uncertainty as to whether the essential role of PDI in the cell is oxidase or isomerase. We have studied the ability of PDI constructs with high oxidase activity and very low isomerase activity to complement the chromosomal deletion of PDI1 in S. cerevisiae. A single catalytic domain of yeast PDI (PDIa') has 50% of the oxidase activity but only 5% of the isomerase activity of wild-type PDI in vitro. Titrating the expression of PDI using the inducible/repressible GAL1-10 promoter shows that the amount of wild-type PDI protein needed to sustain a normal growth rate is 60% or more of the amount normally expressed from the PDI1 chromosomal location. A single catalytic domain (PDIa') is needed in molar amounts that are approximately twice as high as those required for wild-type PDI, which contains two catalytic domains. This comparison suggests that high (>60%) PDI oxidase activity is critical to yeast growth and viability, whereas less than 6% of its isomerase activity is needed.     

Structure prediction and functional analysis of KdsD, an enzyme involved in lipopolysaccharide biosynthesis

Lipopolysaccharide is an essential component of the outer membrane of Gram-negative bacteria and consists of three elements: lipid A, the core oligosaccharide and the O-antigen. The inner core region is highly conserved and contains at least one residue of 3-deoxy-d-manno-octulosonate (Kdo). The first committed step of Kdo biosynthesis is the aldol-keto isomerisation of d-ribulose 5-phosphate to d-arabinose 5-phosphate catalyzed by arabinose 5-phosphate isomerase encoded in Escherichia coli by the kdsD gene.KdsD contains an N-terminal sugar isomerase (SIS) domain commonly found in phosphosugar isomerases but its three-dimensional structure is unknown.The structure of the KdsD SIS domain has been predicted by homology modeling using the hypothetical 3etn protein as a template. Moreover by sequence alignments, comparison with other sugar isomerases structurally related to KdsD, and site-directed mutagenesis we implicated four residues in KdsD activity or substrate recognition. A possible role of these residues in the catalysis is discussed.     

Cyclophilin A interacts with domain II of hepatitis C virus NS5A and stimulates RNA binding in an isomerase-dependent manner

NS5A plays a critical, yet poorly defined, role in hepatitis C virus genome replication. The protein consists of three domains, each of which is able to bind independently to the 3' untranslated region (UTR) of the viral positive strand genomic RNA. The peptidyl-prolyl isomerase cyclophilin A (CypA) binds to domain II, catalyzing cis-trans isomerization. CypA inhibitors such as cyclosporine (CsA) have been shown to inhibit hepatitis C virus (HCV) replication. We show here that CypA stimulated domain II RNA binding activity, and this stimulation was abrogated by CsA. An isomerase mutant of CypA (H126Q) failed to bind to domain II and did not stimulate RNA binding. Finally, we demonstrate that the RNA binding of two domain II mutants, the D316E and D316E/Y317N mutants, previously shown to exhibit CypA independence for RNA replication, was unaffected by CypA. This study provides an insight into the molecular mechanism of CypA activity during HCV replication and further validates the use of CypA inhibitors in HCV therapy.     

Stabilization of recombinant proteins from proteolytic degradation in Escherichia coli using a dual affinity fusion strategy.

A dual affinity fusion approach has been used to study the expression and secretion of labile recombinant proteins in Escherichia coli. Here we show that three small eukaryotic proteins (human proinsulin, a thioredoxin homologous domain of rat protein disulfide isomerase, and the extracellular domain of the alpha 1.2-chain of a human T-cell receptor) are stabilized in vivo using a dual affinity fusion strategy, where the gene encoding the desired product is fused between two genes encoding two different affinity domains. Relatively high yields of full-length product were obtained for all three proteins as compared to when fused to a single fusion partner. Despite the use of a signal peptide, significant amounts of the disulfide protein isomerase and T-cell receptor gene products were maintained in the cytoplasm, while the proinsulin fusion was efficiently secreted to the periplasm. Interestingly, the E. coli heat shock proteins DnaK and GroEL were associated with the fusion proteins isolated from the cytoplasm.     

Transcriptional analysis of catabolite repression in Clostridium acetobutylicum growing on mixtures of D-glucose and D-xylose

Clostridium acetobutylicum is a strict anaerobic organism that is used for biotechnological butanol fermentation. It ferments various hexoses and pentoses to solvents but prefers glucose presumably using a catabolite repression mechanism. Accordingly during growth on a mixture of D-glucose and D-xylose a typical diauxic growth pattern was observed. We used DNA microarrays and real-time RT-PCR to study gene expression during growth on D-glucose, D-xylose mixtures on a defined minimal medium together with monitoring substrate consumption and product formation. We identified two putative operons involved in D-xylose degradation. The first operon (CAC1344-CAC1349) includes a transporter, a xylulose-kinase, a transaldolase, a transketolase, an aldose-1-epimerase and a putative xylose isomerase that has been annotated as an arabinose isomerase. This operon is induced by D-xylose but was catabolite repressed by D-glucose. A second operon (CAC2610-CAC2612) consists of a xylulose-kinase, a hypothetical protein and a gene that has been annotated as a L-fucose isomerase that might in fact code for a xylose isomerase. This operon was induced by D-xylose but was not subject to catabolite repression. In accordance with these results we identified a CRE site in the catabolite repressed operon but not in the operon that was not subject to catabolite repression.     

Isolation and characterization of two tryptophan biosynthetic enzymes, indoleglycerol phosphate synthase and phosphoribosyl anthranilate isomerase, from Bacillus subtilis.

Two of the enzymes responsible for tryptophan biosynthesis in Bacillus subtilis have been extensively purified. These proteins are indole-3-glycerol phosphate synthase and N-(5'-phosphoribosyl) anthranilate isomerase. By comparison to the non-differentiating enteric bacteria in which these two enzymes are fused into a single polypeptide, the isolation of the indoleglycerol phosphate synthase and phosphoribosyl anthranilate isomerase from B. subtilis has demonstrated that the two proteins are separate species in this organism. The two enzymes were clearly separable by anion-exchange chromatography without any significant loss of activity. Molecular weights were determined for both enzymes by gel filtration and sodium dodecyl sulfate-slab gel electrophoresis, and indicated that the indoleglycerol phosphate synthase is the slightly larger of the two proteins. The minimum molecular weight for indoleglycerol phosphate synthase was 23,500, and that for phosphoribosyl anthranilate isomerase was 21,800. Both enzymes have been examined as to conditions necessary to achieve maximal activity of their individual functions and to maintain that activity.     

The structure of yeast enolase at 2.25-A resolution. An 8-fold beta + alpha-barrel with a novel beta beta alpha alpha (beta alpha)6 topology

The three-dimensional structure of yeast enolase has been determined by the multiple isomorphous replacement method followed by the solvent flattening technique. A polypeptide model, corresponding with the known amino acid sequence, has been fitted to the electron density map. Crystallographic restrained least-squares refinement of the model without solvent gave R = 20.0% for 6-2.25-A resolution with good geometry. A model with 182 water molecules and 1 sulfate which is still being refined has presently R = 17.0%. The molecule is a dimer with subunits related by 2-fold crystallographic symmetry. The subunit has dimensions 60 X 55 X 45 A and is built from two domains. The smaller N-terminal domain has an alpha + beta structure based on a three-stranded antiparallel meander and four helices. The main domain is an 8-fold beta + alpha-barrel. The enolase barrel is, however, different from the triose phosphate isomerase barrel; its topology is beta beta alpha alpha (beta alpha)6 rather than (beta alpha)8 as found in triose phosphate isomerase. The inner beta-barrel is not entirely parallel, the second strand is antiparallel to the other strands, and the direction of the first helix is also reversed with respect to the other helices. This supports the hypothesis that some enzymes evolved independently producing the stable structure of beta alpha barrels with either enolase or triose phosphate isomerase topology. The active site of enolase is located at the carboxylic end of the barrel. A fragment of the N-terminal domain and two long loops protruding from the barrel domain form a wide crevice leading to the active site region. Asp246, Glu295, and Asp320 are the ligands of the conformational cation. Other residues in the active site region are Glu168, Asp321, Lys345, and Lys396.     

The crystal structure of human WD40 repeat-containing peptidylprolyl isomerase (PPWD1)

Cyclophilins comprise one of the three classes of peptidylprolyl isomerases found in all eukaryotic and prokaryotic organisms, as well as viruses. Many of the 17 annotated human cyclophilins contain the catalytic domain in tandem with other domains, and many of the specific functions of a particular cyclophilin or its associated domains remain unknown. The structure of the isomerase domain from a spliceosome-associated cyclophilin, PPWD1 (peptidylprolyl isomerase containing WD40 repeat), has been solved to 1.65 A. In the crystal, the N-terminus of one isomerase domain is bound in the active site of a neighboring isomerase molecule in a manner analogous to substrate. NMR solution studies show that this sequence binds to the active site of the cyclophilin, but cannot be turned over by the enzyme. A pseudo-substrate immediately N-terminal to the cyclophilin domain in PPWD1 could have wider implications for the function of this cyclophilin in the spliceosome, where it is located in human cells.     

Isopentenyl pyrophosphate isomerase:dimethylallyl pyrophosphate isomerase: isolation from Claviceps sp. SD 58 and comparison to the mammalian enzyme

Isopentenyl pyrophosphate isomerase:dimethyl pyrophosphate isomerase (EC 5.3.3.2) has been purified to near homogeneity from Claviceps sp. A molecular weight of 35,000 was found by gel exclusion chromatography as well as by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This indicates that the enzyme consists of a single subunit and is in contrast to the Mr 22,000 that we have found for the enzyme from liver. The lability of isomerase from liver, often reported, has been found to be due to its susceptibility to proteolysis. Nine compounds have been tested as inhibitors of both isomerases. The binding of analogs requires the pyrophosphate moiety which may be substituted by a variety of alkyl groups. Inclusion of a polar function in the hydrocarbon portion of the analog greatly reduces interaction with the enzyme. Reversibility of the reaction was not found with a higher homolog of the substrate.     

Structure/function relationships responsible for coenzyme specificity and the isomerase activity of human type 1 3 beta-hydroxysteroid dehydrogenase/isomerase

Human type 1 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD/isomerase) catalyzes the two sequential enzyme reactions on a single protein that converts dehydroepiandrosterone or pregnenolone to androstenedione or progesterone, respectively, in placenta, mammary gland, breast tumors, prostate, prostate tumors, and other peripheral tissues. Our earlier studies show that the two enzyme reactions are linked by the coenzyme product, NADH, of the 3 beta-HSD activity. NADH activates the isomerase activity by inducing a time-dependent conformational change in the enzyme protein. The current study tested the hypothesis that the 3 beta-HSD and isomerase activities shared a common coenzyme domain, and it characterized key amino acids that participated in coenzyme binding and the isomerase reaction. Homology modeling with UDP-galactose-4-epimerase predicts that Asp36 is responsible for the NAD(H) specificity of human 3 beta-HSD/isomerase and identifies the Rossmann-fold coenzyme domain at the amino terminus. The D36A/K37R mutant in the potential coenzyme domain and the D241N, D257L, D258L, and D265N mutants in the potential isomerase domain (previously identified by affinity labeling) were created, expressed, and purified. The D36A/K37R mutant shifts the cofactor preference of both 3 beta-HSD and isomerase from NAD(H) to NADP(H), which shows that the two activities utilize a common coenzyme domain. The D257L and D258L mutations eliminate isomerase activity, whereas the D241N and D265N mutants have nearly full isomerase activity. Kinetic analyses and pH dependence studies showed that either Asp257 or Asp258 plays a catalytic role in the isomerization reaction. These observations further characterize the structure/function relationships of human 3 beta-HSD/isomerase and bring us closer to the goal of selectively inhibiting the type 1 enzyme in placenta (to control the timing of labor) or in hormone-sensitive breast tumors (to slow their growth).     

Binding of FKBP23 to BiP in ER shown by gel filtration chromatography

FKBP23 was found in mouse endoplasmic reticulum (ER) in 1998. It consists of an N-terminal peptidyl-prolyl cis/trans isomerase (PPIase) domain and a C-terminal domain with Ca2+ binding sites. Previously, we reported that FKBP23 specifically binds to BiP, the main protein of the molecular chaperone Hsp70 in ER lumen, and the binding is interrelated with the Ca2+ concentration. In this work we have found the existence of the complex FKBP23/BiP by separation of an ER extract using gel filtration chromatography (GFC), and that the existence of this complex is Ca2+-interrelated. This result further verified the Ca2+-interrelated binding of these two proteins in vivo.     

Mutations of the membrane-bound disulfide reductase DsbD that block electron transfer steps from cytoplasm to periplasm in Escherichia coli

The cytoplasmic membrane protein DsbD keeps the periplasmic disulfide isomerase DsbC reduced, using the cytoplasmic reducing power of thioredoxin. DsbD contains three domains, each containing two reactive cysteines. One membrane-embedded domain, DsbDbeta, transfers electrons from thioredoxin to the carboxy-terminal thioredoxin-like periplasmic domain DsbDgamma. To evaluate the role of conserved amino acid residues in DsbDbeta in the electron transfer process, we substituted alanines for each of 19 conserved amino acid residues and assessed the in vivo redox states of DsbC and DsbD. The mutant DsbDs of 11 mutants which caused defects in DsbC reduction showed relatively oxidized redox states. To analyze the redox state of each DsbD domain, we constructed a thrombin-cleavable DsbD (DsbDTH) from which we could generate all three domains as separate polypeptide chains by thrombin treatment in vitro. We divided the mutants with strong defects into two classes. The first mutant class consists of mutant DsbDbeta proteins that cannot receive electrons from cytoplasmic thioredoxin, resulting in a DsbD that has all six of its cysteines disulfide bonded. The second mutant class represents proteins in which the transfer of electrons from DsbDbeta to DsbDgamma appears to be blocked. This class includes the mutant with the most clear-cut defect, P284A. We relate the properties of the mutants to the positions of the amino acids in the structure of DsbD and discuss mechanisms that would interfere with the electron transfer process.