Analysis of Amino Acid Residues Involved in Catalysis of Polyethylene Glycol Dehydrogenase from Sphingopyxis terrae, Using Three-Dimensional Molecular Modeling-Based Kinetic Characterization of Mutants

Polyethylene glycol dehydrogenase (PEGDH) from Sphingopyxis terrae (formerly Sphingomonas terrae) is composed of 535 amino acid residues and one flavin adenine dinucleotide per monomer protein in a homodimeric structure. Its amino acid sequence shows 28.5 to 30.5% identity with glucose oxidases from Aspergillus niger and Penicillium amagasakiense. The ADP-binding site and the signature 1 and 2 consensus sequences of glucose-methanol-choline oxidoreductases are present in PEGDH. Based on three-dimensional molecular modeling and kinetic characterization of wild-type PEGDH and mutant PEGDHs constructed by site-directed mutagenesis, residues potentially involved in catalysis and substrate binding were found in the vicinity of the flavin ring. The catalytically important active sites were assigned to His-467 and Asn-511. One disulfide bridge between Cys-379 and Cys-382 existed in PEGDH and seemed to play roles in both substrate binding and electron mediation. The Cys-297 mutant showed decreased activity, suggesting the residue's importance in both substrate binding and electron mediation, as well as Cys-379 and Cys-382. PEGDH also contains a motif of a ubiquinone-binding site, and coenzyme Q₁₀ was utilized as an electron acceptor. Thus, we propose several important amino acid residues involved in the electron transfer pathway from the substrate to ubiquinone.     

Identification of essential amino acid residues in valine dehydrogenase from Streptomyces albus

Cys-29 and Cys-251 of Streptomyces albus valine dehydrogenase (ValDH) were highly conserved in the corresponding region of NAD(P)(+)-dependent amino acid dehydroganase sequences. To ascertain the functional role of these cysteine residues in S. albus ValDH, site-directed mutagenesis was performed to change each of the two residues to serine. Kinetic analyses of the enzymes mutated at Cys-29 and Cys-251 revealed that these residues are involved in catalysis. We also constructed mutant ValDH by substituting valine for leucine at 305 by site-directed mutagenesis. This residue was chosen, because it has been proposed to be important for substrate discrimination by phenylalanine dehydrogenase (PheDH) and leucine dehydrogenase (LeuDH). Kinetic analysis of the V305L mutant enzyme revealed that it is involved in the substrate binding site. However it displayed less activity than the wild type enzyme toward all aliphatic and aromatic amino acids tested.     

Residue cysteine 232 is important for substrate transport of neutral amino acid transporter,SNAT4

SNAT4 is a system A type amino acid transporter that primarily expresses in liver and mediates the transport of L-alanine. To determine the critical amino acid residue(s) involved in substrate transport function of SNAT4, we used hydrosulfate cross-linking MTS reagents - MMTS and MTSEA. These two reagents caused inhibition of L-alanine transport by wild-type SNAT4. There are 5 cysteine residues in SNAT4 and among them; residues Cys-232 and Cys-345 are located in the transmembrane domains. Mutation of Cys-232, but not Cys-345, inhibited transport function of SNAT4 and also rendered SNAT4 less sensitive to the cross-linking by MMTS and MTSEA. The results suggested that TMD located Cys-232 is an aqueous accessible residue, likely to be located close to the core of substrate binding site. Mutation of Cys-232 to serine similarly attenuated the transport of L-alanine substrate. Biotinylation analysis showed that C232A mutant of SNAT4 was equally capable as wild-type SNAT4 of expressing on the cell surface. Moreover, single site mutant, C232A was also found to be more resistant to MTS inhibition than double mutant C18A,C345A, further confirming the aqueous accessibility of Cys-232 residue. We also showed that mutation of Cys-232 to alanine reduced the maximal velocity (Vmax), but had minimal effect on binding affinity (Km). Together, these data suggest that residue Cys-232 at 4^th transmembrane domain of SNAT4 has a major influence on substrate transport capacity, but not on substrate binding affinity.     

Identification of functionally relevant residues of the rat ileal apical sodium-dependent bile acid cotransporter

The mechanisms underlying the transport of bile acids by apical sodium-dependent bile acid transporter (Asbt) are not well defined. To further identify the functionally relevant residues, thirteen conserved negatively (Asp and Glu) and positively (Lys and Arg) charged residues plus Cys-270 of rat Asbt were replaced with Ala or Gln by site-directed mutagenesis. Seven of the fourteen residues of rat Asbt were identified as functionally important by taurocholate transport studies, substrate inhibition assays, confocal microscopy, and electrophysiological methods. The results showed that Asp-122, Lys-191, Lys-225, Lys-256, Glu-261, and Lys-312,Lys-313 residues of rat Asbt are critical for transport function and may determine substrate specificity. Arg-64 may be located at a different binding site to assist in interaction with non-bile acid organic anions. For bile acid transport by Asbt, Na(+) ion movement is a voltage-dependent process that tightly companied with taurocholate movement. Asp-122 and Glu-261 play a critical role in the interaction of a Na(+) ion and ligand with Asbt. Cys-270 is not essential for the transport process. These studies provide new details about the amino acid residues of Asbt involved in binding and transport of bile acids and Na(+).     

Biochemistry of terminal deoxynucleotidyltransferase. Affinity labeling and identification of the deoxynucleoside triphosphate binding domain of terminal deoxynucleotidyltransferase

Using the technique of UV-mediated cross-linking of nucleotides to their acceptor sites (Modak, M. J., and Gillerman-Cox, E. (1982) J. Biol. Chem. 257, 15105-15109), we have labeled calf terminal deoxynucleotidyltransferase (TdT) with [32P]dTTP. The specificity of dTTP cross-linking at the substrate binding site in TdT is demonstrated by the competitive inhibition of the cross-linking reaction by other deoxynucleoside triphosphates, and ATP and its analogues, requiring concentrations consistent with their kinetic constants. Tryptic peptide mapping of the [32P]dTTP-labeled enzyme showed the presence of a single radioactive peptide fraction that contained the site of dTTP cross-linking. The amino acid composition and sequence analysis of the radioactive peptide fraction revealed it to contain two tryptic peptides, spanning residues 221-231 and 234-249. Since these two peptides were covalently linked to dTTP, the region encompassed by them constitutes a substrate binding domain in TdT. Further proteolytic digestion of the tryptic peptide-dTTP complex, using V8 protease, yielded a smaller peptide, and its analysis narrowed the substrate binding domain to 14 amino acids corresponding to residues 224-237 in the primary amino acid sequence of TdT. Furthermore, 2 cysteine residues, Cys-227 and Cys-234, within this domain were found to be involved in the cross-linking of dTTP, suggesting their participation in the process of substrate binding in TdT.     

Crystal structure of red sea bream transglutaminase

The crystal structure of the tissue-type transglutaminase from red sea bream liver (fish-derived transglutaminase, FTG) has been determined at 2.5-A resolution using the molecular replacement method, based on the crystal structure of human blood coagulation factor XIII, which is a transglutaminase zymogen. The model contains 666 residues of a total of 695 residues, 382 water molecules, and 1 sulfate ion. FTG consists of four domains, and its overall and active site structures are similar to those of human factor XIII. However, significant structural differences are observed in both the acyl donor and acyl acceptor binding sites, which account for the difference in substrate preferences. The active site of the enzyme is inaccessible to the solvent, because the catalytic Cys-272 hydrogen-bonds to Tyr-515, which is thought to be displaced upon acyl donor binding to FTG. It is postulated that the binding of an inappropriate substrate to FTG would lead to inactivation of the enzyme because of the formation of a new disulfide bridge between Cys-272 and the adjacent Cys-333 immediately after the displacement of Tyr-515. Considering the mutational studies previously reported on the tissue-type transglutaminases, we propose that Cys-333 and Tyr-515 are important in strictly controlling the enzymatic activity of FTG.     

Alteration of substrate specificity of cholesterol oxidase from Streptomyces sp. by site-directed mutagenesis

Despite the structural similarities between cholesterol oxidase from Streptomyces and that from Brevibacterium, both enzymes exhibit different characteristics, such as catalytic activity, optimum pH and temperature. In attempts to define the molecular basis of differences in catalytic activity or stability, substitutions at six amino acid residues were introduced into cholesterol oxidase using site-directed mutagenesis of its gene. The amino acid substitutions chosen were based on structural comparisons of cholesterol oxidases from Streptomyces and BREVIBACTERIUM: Seven mutant enzymes were constructed with the following amino acid substitutions: L117P, L119A, L119F, V145Q, Q286R, P357N and S379T. All the mutant enzymes exhibited activity with the exception of that with the L117P mutation. The resulting V145Q mutant enzyme has low activities for all substrates examined and the S379T mutant enzyme showed markedly altered substrate specificity compared with the wild-type enzyme. To evaluate the role of V145 and S379 residues in the reaction, mutants with two additional substitutions in V145 and four in S379 were constructed. The mutant enzymes created by the replacement of V145 by Asp and Glu had much lower catalytic efficiency for cholesterol and pregnenolone as substrates than the wild-type enzyme. From previous studies and this study, the V145 residue seems to be important for the stability and substrate binding of the cholesterol oxidase. In contrast, the catalytic efficiencies (k(cat)/K(m)) of the S379T mutant enzyme for cholesterol and pregnenolone were 1.8- and 6.0-fold higher, respectively, than those of the wild-type enzyme. The enhanced catalytic efficiency of the S379T mutant enzyme for pregnenolone was due to a slightly high k(cat) value and a low K(m) value. These findings will provide several ideas for the design of more powerful enzymes that can be applied to clinical determination of serum cholesterol levels and as sterol probes.     

Functional role of the "aromatic cage" in human monoamine oxidase B: structures and catalytic properties of Tyr435 mutant proteins

Current structural results of several flavin-dependent amine oxidizing enzymes including human monoamine oxidases A and B (MAO A and MAO B) show aromatic amino acid residues oriented approximately perpendicular to the flavin ring, suggesting a functional role in catalysis. In the case of human MAO B, two tyrosyl residues (Y398 and Y435) are found in the substrate binding site on the re face of the covalent flavin ring [Binda et al. (2002) J. Biol. Chem. 277, 23973-23976]. To probe the functional significance of this structure, Tyr435 in MAO B was mutated with the amino acids Phe, His, Leu, or Trp, the mutant proteins expressed in Pichia pastoris, and purified to homogeneity. Each mutant protein contains covalent FAD and exhibits a high level of catalytic functionality. No major alterations in active site structures are detected on comparison of their respective crystal structures with that of WT enzyme. The relative k(cat)/K(m) values for each mutant enzyme show Y435 > Y435F = Y435L = Y435H > Y435W. A similar behavior is also observed with the membrane-bound forms of MAO A and MAO B (MAO A Y444 mutant enzymes are found to be unstable on membrane extraction). p-Nitrobenzylamine is found to be a poor substrate while p-nitrophenethylamine is found to be a good substrate for all WT and mutant forms of MAO B. Analysis of these kinetic and structural data suggests the function of the "aromatic cage" in MAO to include a steric role in substrate binding and access to the flavin coenzyme and to increase the nucleophilicity of the substrate amine moiety. These results are consistent with a proposed polar nucleophilic mechanism for catalytic amine oxidation.     

Structural basis of free reduced flavin generation by flavin reductase from Thermus thermophilus HB8

Free reduced flavins are involved in a variety of biological functions. They are generated from NAD(P)H by flavin reductase via co-factor flavin bound to the enzyme. Although recent findings on the structure and function of flavin reductase provide new information about co-factor FAD and substrate NAD, there have been no reports on the substrate flavin binding site. Here we report the structure of TTHA0420 from Thermus thermophilus HB8, which belongs to flavin reductase, and describe the dual binding mode of the substrate and co-factor flavins. We also report that TTHA0420 has not only the flavin reductase motif GDH but also a specific motif YGG in C terminus as well as Phe-41 and Arg-11, which are conserved in its subclass. From the structure, these motifs are important for the substrate flavin binding. On the contrary, the C terminus is stacked on the NADH binding site, apparently to block NADH binding to the active site. To identify the function of the C-terminal region, we designed and expressed a mutant TTHA0420 enzyme in which the C-terminal five residues were deleted (TTHA0420-ΔC5). Notably, the activity of TTHA0420-ΔC5 was about 10 times higher than that of the wild-type enzyme at 20-40 °C. Our findings suggest that the C-terminal region of TTHA0420 may regulate the alternative binding of NADH and substrate flavin to the enzyme.     

Functional analysis by site-directed mutagenesis of individual amino acid residues in the flavin domain of Neurospora crassa nitrate reductase

Nitrate reductase of Neurospora crassa is a complex multi-redox protein composed of two identical subunits, each of which contains three distinct domains, an amino-terminal domain that contains a molybdopterin cofactor, a central heme-containing domain, and a carboxy-terminal domain which binds a flavin and a pyridine nucleotide cofactor. The flavin domain of nitrate reductase appears to have structural and functional similarity to ferredoxin NADPH reductase (FNR). Using the crystal structure of FNR and amino acid identities in numerous nitrate reductases as guides, site-directed mutagenesis was used to replace specific amino acids suspected to be involved in the binding of the flavin or pyridine nucleotide cofactors and thus important for the catalytic function of the flavin domain. Each mutant flavin domain protein was expressed in Escherichia coli and analyzed for NADPH: ferricyanide reductase activity. The effect of each amino acid substitution upon the activity of the complete nitrate reductase reaction was also examined by transforming each manipulated gene into a nit-3 ^? null mutant of N. crassa. Our results identify amino acid residues which are critical for function of the flavin domain of nitrate reductase and appear to be important for the binding of the flavin or the pyridine nucleotide cofactors.     

Functional analysis by site-directed mutagenesis of individual amino acid residues in the flavin domain of Neurospora crassa nitrate reductase

Nitrate reductase of Neurospora crassa is a complex multi-redox protein composed of two identical subunits, each of which contains three distinct domains, an amino-terminal domain that contains a molybdopterin cofactor, a central heme-containing domain, and a carboxy-terminal domain which binds a flavin and a pyridine nucleotide cofactor. The flavin domain of nitrate reductase appears to have structural and functional similarity to ferredoxin NADPH reductase (FNR). Using the crystal structure of FNR and amino acid identities in numerous nitrate reductases as guides, site-directed mutagenesis was used to replace specific amino acids suspected to be involved in the binding of the flavin or pyridine nucleotide cofactors and thus important for the catalytic function of the flavin domain. Each mutant flavin domain protein was expressed in Escherichia coli and analyzed for NADPH: ferricyanide reductase activity. The effect of each amino acid substitution upon the activity of the complete nitrate reductase reaction was also examined by transforming each manipulated gene into a nit-3 ^– null mutant of N. crassa. Our results identify amino acid residues which are critical for function of the flavin domain of nitrate reductase and appear to be important for the binding of the flavin or the pyridine nucleotide cofactors.     

Site-directed mutagenesis of the N-terminal region of IGF binding protein 1; analysis of IGF binding capability.

To define domains involved in IGF binding 60 N-terminal amino acid residues of IGFBP-1 were deleted. This deletion resulted in loss of IGF binding suggesting that the N-terminus may enclose an IGF binding domain. However, most point mutations introduced in this region did not affect IGF binding. In contrast to Cys-34, only substitution of Cys-38 for a tyrosine residue abolished IGF binding. With the determination that all 18 cysteine residues are involved in disulphide bond formation our data suggest that, although not all cysteines contribute to the same extent, the ligand binding site may be spatially organized.     

The active site of hydroxynitrile lyase from Prunus amygdalus: Modeling studies provide new insights into the mechanism of cyanogenesis

The FAD-dependent hydroxynitrile lyase from almond (Prunus amygdalus, PaHNL) catalyzes the cleavage of R-mandelonitrile into benzaldehyde and hydrocyanic acid. Catalysis of the reverse reaction-the enantiospecific formation of alpha-hydroxynitriles--is now widely utilized in organic syntheses as one of the few industrially relevant examples of enzyme-mediated C-C bond formation. Starting from the recently determined X-ray crystal structure, systematic docking calculations with the natural substrate were used to locate the active site of the enzyme and to identify amino acid residues involved in substrate binding and catalysis. Analysis of the modeled substrate complexes supports an enzymatic mechanism that includes the flavin cofactor as a mere "spectator" of the reaction and relies on general acid/base catalysis by the conserved His-497. Stabilization of the negative charge of the cyanide ion is accomplished by a pronounced positive electrostatic potential at the binding site. PaHNL activity requires the FAD cofactor to be bound in its oxidized form, and calculations of the pKa of enzyme-bound HCN showed that the observed inactivation upon cofactor reduction is largely caused by the reversal of the electrostatic potential within the active site. The suggested mechanism closely resembles the one proposed for the FAD-independent, and structurally unrelated HNL from Hevea brasiliensis. Although the actual amino acid residues involved in the catalytic cycle are completely different in the two enzymes, a common motif for the mechanism of cyanogenesis (general acid/base catalysis plus electrostatic stabilization of the cyanide ion) becomes evident.     

A novel approach to the characterization of substrate specificity in short/branched chain Acyl-CoA dehydrogenase

Rat and human short/branched chain acyl-CoA dehydrogenases exhibit key differences in substrate specificity despite an overall amino acid identity of 85% between them. Rat short/branched chain acyl-CoA dehydrogenases (SBCAD) are more active toward substrates with longer carbon side chains than human SBCAD, whereas the human enzyme utilizes substrates with longer primary carbon chains. The mechanism underlying this difference in substrate specificity was investigated with a novel surface plasmon resonance assay combined with absorbance and circular dichroism spectroscopy, and kinetics analysis of wild type SBCADs and mutants with altered amino acid residues in the substrate binding pocket. Results show that a relatively few amino acid residues are critical for determining the difference in substrate specificity seen between the human and rat enzymes and that alteration of these residues influences different portions of the enzyme mechanism. Molecular modeling of the SBCAD structure suggests that position 104 at the bottom of the substrate binding pocket is important in determining the length of the primary carbon chain that can be accommodated. Conformational changes caused by alteration of residues at positions 105 and 177 directly affect the rate of electron transfer in the dehydrogenation reactions, and are likely transmitted from the bottom of the substrate binding pocket to beta-sheet 3. Differences between the rat and human enzyme at positions 383, 222, and 220 alter substrate specificity without affecting substrate binding. Modeling predicts that these residues combine to determine the distance between the flavin ring of FAD and the catalytic base, without changing the opening of the substrate binding pocket.     

唾液乳杆菌BSH1及其突变体的底物特异性

为了解析胆盐水解酶催化中心中关键氨基酸位点与其底物特异性的关系,以大肠杆菌pET-20b(+)表达系统为分子改造平台,采用理性设计,结合氨基酸定点突变的方法,成功构建了唾液乳杆菌Lactobacillus salivarius胆盐水解酶BSH1的7种突变体。通过对比L.salivarius BSH1及其突变体对6种结合胆盐的底物特异性表明,7种突变体对不同的结合胆盐的水解活性有所改变。结果说明,Cys2和Thr264分别是BSH1催化TCA和GCA的关键残基,且对酶的催化活性的保持具有关键作用。其中,高保守性的氨基酸位点Cys2不是BSH1唯一的活性位点,而其他突变的氨基酸位点可能作为BSH1的结合位点参与了底物的结合,也可能影响了底物进入BSH1活性中心的通道或底物结合口袋的体积与形状,进而影响了BSH1对不同结合胆盐的水解活性。     

Structural and biochemical characterization of recombinant wild type and a C30A mutant of trimethylamine dehydrogenase from methylophilus methylotrophus (sp. W(3)A(1))

Trimethylamine dehydrogenase (TMADH) is an iron-sulfur flavoprotein that catalyzes the oxidative demethylation of trimethylamine to form dimethylamine and formaldehyde. It contains a unique flavin, in the form of a 6-S-cysteinyl FMN, which is bent by approximately 25 degrees along the N5-N10 axis of the flavin isoalloxazine ring. This unusual conformation is thought to modulate the properties of the flavin to facilitate catalysis, and has been postulated to be the result of covalent linkage to Cys-30 at the flavin C6 atom. We report here the crystal structures of recombinant wild-type and the C30A mutant TMADH enzymes, both determined at 2.2 A resolution. Combined crystallographic and NMR studies reveal the presence of inorganic phosphate in the FMN binding site in the deflavo fraction of both recombinant wild-type and C30A proteins. The presence of tightly bound inorganic phosphate in the recombinant enzymes explains the inability to reconstitute the deflavo forms of the recombinant wild-type and C30A enzymes that are generated in vivo. The active site structure and flavin conformation in C30A TMADH are identical to those in recombinant and native TMADH, thus revealing that, contrary to expectation, the 6-S-cysteinyl FMN link is not responsible for the 25 degrees butterfly bending along the N5-N10 axis of the flavin in TMADH. Computational quantum chemistry studies strongly support the proposed role of the butterfly bend in modulating the redox properties of the flavin. Solution studies reveal major differences in the kinetic behavior of the wild-type and C30A proteins. Computational studies reveal a hitherto, unrecognized, contribution made by the S(gamma) atom of Cys-30 to substrate binding, and a role for Cys-30 in the optimal geometrical alignment of substrate with the 6-S-cysteinyl FMN in the enzyme active site.     

Bacillus subtilis mutant succinate dehydrogenase lacking covalently bound flavin: identification of the primary defect and studies on the iron-sulfur clusters in mutated and wild-type enzyme

Succinate dehydrogenase consists of two protein subunits and contains one FAD and three iron-sulfur clusters. The flavin is covalently bound to a histidine in the larger, Fp, subunit. The reduction oxidation midpoint potentials of the clusters designated S-1, S-2, and S-3 in Bacillus subtilis wild-type membrane-bound enzyme were determined as +80, -240, and -25 mV, respectively. Magnetic spin interactions between clusters S-1 and S-2 and between S-1 and S-3 were detected by using EPR spectroscopy. The point mutations of four B. subtilis mutants with defective Fp subunits were mapped. The gene of the mutant specifically lacking covalently bound flavin in the enzyme was cloned. The mutation was determined from the DNA sequence as a glycine to aspartate substitution at a conserved site seven residues downstream from the histidine that binds the flavin in wild-type enzyme. The redox midpoint potential of the iron-sulfur clusters and the magnetic spin interactions in mutated succinate dehydrogenases were indistinguishable from the those of the wild type. This shows that flavin has no role in the measured magnetic spin interactions or in the structure and stability of the iron-sulfur clusters. It is concluded from sequence and mutant studies that conserved amino acid residues around the histidyl-FAD are important for FAD binding; however, amino acids located more than 100 residues downstream from the histidyl in the Fp subunit can also effect flavinylation.     

Roles of disulfide linkage and calcium ion-mediated interactions in assembly and disassembly of virus-like particles composed of simian virus 40 VP1 capsid protein

The simian virus 40 capsid is composed of 72 pentamers of VP1 protein. Although the capsid is known to dissociate to pentamers in vitro following simultaneous treatment with reducing and chelating agents, the functional roles of disulfide linkage and calcium ion-mediated interactions are not clear. To elucidate the roles of these interactions, we introduced amino acid substitutions in VP1 at cysteine residues and at residues involved in calcium binding. We expressed the mutant proteins in a baculovirus system and analyzed both their assembly into virus-like particles (VLPs) in insect cells and the disassembly of those VLPs in vitro. We found that disulfide linkages at both Cys-9 and Cys-104 conferred resistance to proteinase K digestion on VLPs, although neither linkage was essential for the formation of VLPs in insect cells. In particular, reduction of the disulfide linkage at Cys-9 was found to be critical for VLP dissociation to VP1 pentamers in the absence of calcium ions, indicating that disulfide linkage at Cys-9 prevents VLP dissociation, probably by increasing the stability of calcium ion binding. We found that amino acid substitutions at carboxy-terminal calcium ion binding sites (Glu-329, Glu-330, and Asp-345) resulted in the frequent formation of unusual tubular particles as well as VLPs in insect cells, indicating that these residues affect the accuracy of capsid assembly. In addition, unexpectedly, amino acid substitutions at any of the calcium ion binding sites tested, especially at Glu-157, resulted in increased stability of VLPs in the absence of calcium ions in vitro. These results suggest that appropriate affinities of calcium ion binding are responsible for both assembly and disassembly of the capsid.     

Crystal structure of the cytochrome p450cam mutant that exhibits the same spectral perturbations induced by putidaredoxin binding

The cytochrome P450cam active site is known to be perturbed by binding to its redox partner, putidaredoxin (Pdx). Pdx binding also enhances the camphor monooxygenation reaction (Nagano, S., Shimada, H., Tarumi, A., Hishiki, T., Kimata-Ariga, Y., Egawa, T., Suematsu, M., Park, S.-Y., Adachi, S., Shiro, Y., and Ishimura, Y. (2003) Biochemistry 42, 14507-14514). These effects are unique to Pdx because nonphysiological electron donors are unable to support camphor monooxygenation. The accompanying 1H NMR paper (Tosha, T., Yoshioka, S., Ishimori, K., and Morishima, I. (2004) J. Biol. Chem. 279, 42836-42843) shows that the conformation of active site residues, Thr-252 and Cys-357, and the substrate in the ferrous (Fe(II)) CO complex of the L358P mutant mimics that of the wild-type enzyme complexed to Pdx. To explore how these changes are transmitted from the Pdx-binding site to the active site, we have solved the crystal structures of the ferrous and ferrous-CO complex of wild-type and the L358P mutant. Comparison of these structures shows that the L358P mutation results in the movement of Arg-112, a residue known to be important for putidaredoxin binding, toward the heme. This change could optimize the Pdx-binding site leading to a higher affinity for Pdx. The mutation also pushes the heme toward the substrate and ligand binding pocket, which relocates the substrate to a position favorable for regio-selective hydroxylation. The camphor is held more firmly in place as indicated by a lower average temperature factor. Residues involved in the catalytically important proton shuttle system in the I helix are also altered by the mutation. Such conformational alterations and the enhanced reactivity of the mutant oxy complex with non-physiological electron donors suggest that Pdx binding optimizes the distal pocket for monooxygenation of camphor.