Shin-ichi Ozaki

Effects of the Location of Distal Histidine in the Reaction of Myoglobin with Hydrogen Peroxide

To clarify how the location of distal histidine affects the activation process of H2O2 by heme proteins, we have characterized reactions with H2O2 for the L29H/H64L and F43H/H64L mutants of sperm whale myoglobin (Mb), designed to locate the histidine farther from the heme iron. Whereas the L29H/H64L double substitution retarded the reaction with H2O2, an 11-fold rate increase versus wild-type Mb was observed for the F43H/H64L mutant. The V max values for 1-electron oxidations by the myoglobins correlate well with the varied reactivities with H2O2. The functions of the distal histidine as a general acid-base catalyst were examined based on the reactions with cumene hydroperoxide and cyanide, and only the histidine in F43H/H64L Mb was suggested to facilitate heterolysis of the peroxide bond. The x-ray crystal structures of the mutants confirmed that the distal histidines in F43H/H64L Mb and peroxidase are similar in distance from the heme iron, whereas the distal histidine in L29H/H64L Mb is located too far to enhance heterolysis. Our results indicate that the proper positioning of the distal histidine is essential for the activation of H2O2 by heme enzymes.

Biosynthesis of vitamin B12: Discovery of the enzymes for oxidative ring contraction and insertion of the fourth methyl group

In the vitamin B12 biosynthetic pathway the enzymes responsible for the conversion of precorrin‐3 to precorrin‐4 have been identified as the gene products of cobG and cobJ from Pseudomonas denitrificans. CobG catalyzes the oxidation of precorrin‐3 to precorrin‐3x (a hydroxy lactone) whereas CobJ is a SAM‐dependent C‐17 methyl transferase and is necessary for ring contraction. A mechanism for ring contraction is proposed.

Rational molecular design of a catalytic site: engineering of catalytic functions to the myoglobin active site framework

Abstract Proteins that contain the heme prosthetic group are responsible for many different types of catalytic activity. Understanding the mechanisms through which a particular type of catalytic activity is favored over the others remains a significant challenge. Recently, the most common strategy for structure–function studies for a particular enzyme has involved substitution of amino acid residues by site-directed mutagenesis followed by investigations of the effect of the substitution on the catalytic activity of that system. This work describes a significant departure from this common strategy. Instead, we seek to convert a non-enzymatic hemoprotein into one that is capable of catalytic activity. In so doing, we expect to gain an understanding of the general structural requirements for particular enzymatic functions. Comparison of X-ray crystal structures of myoglobin and peroxidases reveals differences in arrangement of amino acid residues in the heme pockets. On the basis of these structural differences and the reaction mechanism of peroxidases, we have rationally designed several myoglobin mutants in order to convert myoglobin into a peroxidase-like enzyme. We have discovered that the location of the distal histidine in the active site provides a critical balance between the formation and subsequent decay of the oxo-ferryl porphyrin radical cation (compound I), a catalytic species for one- and two-electron oxidation and oxygen transfer reactions. The mutants prepared in this work have been altered in such a manner that they have permitted compound I to be observed in myoglobin for the first time. This allows us to investigate mechanistic details under single turnover conditions by use of double mixing stopped-flow spectroscopy. Furthermore, some of the mutants we have constructed might be useful as good catalysts for asymmetric oxidations. In this short review, we describe our attempts to elucidate structure–function relationships on the activation of the oxygen–oxygen bond of peroxides by hemoproteins.

Asymmetric oxidation catalyzed by myoglobin mutants

Abstract The sperm whale myoglobin active site mutants (L29H/H64L and F43H/H64L Mb) have been shown to catalyze the asymmetric oxidation of sulfides and olefins. Thioanisole, ethyl phenyl sulfide, and cis -β-methylstyrene are oxidized by L29H/H64L Mb with more than 95% enantiomeric excess (% ee). On the other hand, the F43H/H64L mutant transforms trans -β-methylstyrene into the trans -epoxide with 96% ee. The dominant sulfoxide product in the incubation of alkyl phenyl thioethers is the R isomer; however, the mutants afford dominantly the S isomer of aromatic bicyclic sulfoxides. The results help us to rationalize the difference in the preferred stereochemistry of the Mb mutant-catalyzed reactions. Furthermore, the Mb mutants exhibit an improvement in the oxidation rate up to 300-fold with respect to wild type.

Expression of 9 Salmonella typhimurium enzymes for cobinamide synthesis Identification of the 11-methyl and 20-methyl transferases of corrin biosynthesis

Nine of the cbi genes from the 17.5 kb cob operon of Salmonella typhimurium previously shown by genetic studies to be involved in the biosynthesis of cobinamide from precorrin‐2, have been subcloned and expressed in Escherichia coli. Seven of the gene products were found in the soluble fraction of cell lysates and have been purified. The gene products corresponding to cbi E, F, H and L were shown by SAM binding and by homology with other SAM‐binding proteins to be candidates for the methyltransferases of vitamin B12 biosynthesis. The enzymatic functions of the gene products of cbiL and cbiF are associated with C‐methylation at C‐20 of precorrin‐2 and C‐11 of precorrin‐3.

Regioselective glucosidation of trans-resveratrol in Escherichia coli expressing glucosyltransferase from Phytolacca americana

A glucosyltransferase (GT) of Phytolacca americana (PaGT3) was expressed in Escherichia coli and purified for the synthesis of two O-β-glucoside products of trans-resveratrol. The reaction was moderately regioselective with a ratio of 4′-O-β-glucoside: 3-O-β-glucoside at 10:3. We used not only the purified enzyme but also the E. coli cells containing the PaGT3 gene for the synthesis of glycoconjugates. E. coli cell cultures also have other advantages, such as a shorter incubation time compared with cultured plant cells, no need for the addition of exogenous glucosyl donor compounds such as UDP-glucose, and almost complete conversion of the aglycone to the glucoside products. Furthermore, a homology model of PaGT3 and mutagenesis studies suggested that His-20 would be a catalytically important residue.

Inhibition of Heme Uptake in Pseudomonas aeruginosa by its Hemophore (HasAp ) Bound to Synthetic Metal Complexes

The heme acquisition system A protein secreted by Pseudomonas aeruginosa (HasA(p)) can capture several synthetic metal complexes other than heme. The crystal structures of HasA(p) harboring synthetic metal complexes revealed only small perturbation of the overall HasA(p) structure. An inhibitory effect upon heme acquisition by HasA(p) bearing synthetic metal complexes was examined by monitoring the growth of Pseudomonas aeruginosa PAO1. HasA(p) bound to iron-phthalocyanine inhibits heme acquisition in the presence of heme-bound HasA(p) as an iron source.

Spectroscopic studies on HasA from Yersinia pseudotuberculosis

Heme acquisition system A (HasA) is known as a hemophore in Gram-negative pathogens. The ferric heme iron is coordinated by Tyr-75 and His-32 in holo-HasA from Pseudomonas aeruginosa (HasApa). In contrast, in holo-HasA from Yersinia pseudotuberculosis (HasAyp), our spectroscopic studies suggest that only Tyr-75 coordinates to the ferric heme iron. The substitution of Gln-32 with alanine in HasAyp does not alter the spectroscopic properties, indicating that Gln-32 is not an axial ligand for the heme iron. Somewhat surprisingly, the Y75A mutant of HasAyp can capture a free hemin molecule but the rate of hemin uptake is slower than that of wild type, suggesting that the hydrophobic interaction in the heme pocket may also play a role in heme acquisition. Unlike in wild type apoprotein, ferric heme transfer from Hb to Y75A apo-HasAyp has not been observed. These results imply that coordination (bonding/interaction) between Tyr-75 and the heme iron is important for heme transfer from Hb. Interestingly, HasAyp differs from HasApa in its ability to bind the ferrous heme iron. Apo-HasAyp can capture ferrous heme and resonance Raman spectra of ferrous-carbon monoxide holo-HasAyp suggest that Tyr-75 is protonated when the heme iron is in the ferrous state. The ability of HasAyp to acquire the ferrous heme iron might be beneficial to Y. pseudotuberculosis, a facultative anaerobe in the Enterobacteriaceae family.

The crystal structure of heme acquisition system A from Yersinia pseudotuberculosis (HasAypt): Roles of the axial ligand Tyr75 and two distal arginines in heme binding

Some Gram-negative pathogens utilize an extracellular heme-binding protein called hemophore to satisfy their needs for iron, a metal element essential for most living things. We report here crystal structures of heme acquisition system A from Yersinia pseudotuberculosis (HasAypt) and its Y75A mutant. The wild-type HasAypt structure revealed that the heme iron is coordinated with Tyr75 and a water molecule. The heme-bound water molecule makes extensive hydrogen bond network that includes Arg40 and Arg144 on the distal heme pocket. Arg40, highly conserved for HasAs from Yersinia species, forms a salt bridge with the propionate side chain of heme, and makes π-π stacking and hydrophobic interactions with porphyrin plane. Interestingly, similar Arg-heme interactions are also found for periplasmic heme transporter, PhuT, suggesting that this is an example of a convergent evolution and one of the important interactions for bacterial heme transportation. Heme titration, heme binding kinetics, and the crystal structures of wild-type and Y75A proteins show that, although Tyr75 is primarily important for heme capturing, other interactions with Arg40, Arg144, and hydrophobic residues also contribute for heme acquisition. We also found that HasAypt can form a dimer in solution. The structure of the domain-swapped Y75A HasAypt dimer shows the presence of two low-spin heme molecules coordinated with His84 and His140, and displacement of the Arg40 loop of dimeric Y75A HasAypt results in deformation of the heme-binding pocket. A similar rearrangement of the distal heme loop might occur in heme transfer from HasAypt to HasRypt.

HmuS from Yersinia pseudotuberculosis is a non-canonical heme-degrading enzyme to acquire iron from heme

Some Gram-negative pathogens import host heme into the cytoplasm and utilize it as an iron source for their survival. We report here that HmuS, encoded by the heme utilizing system (hmu) locus, cleaves the protoporphyrin ring to release iron from heme. A liquid chromatography/mass spectrometry analysis revealed that the degradation products of this reaction are two biliverdin isomers that result from transformation of a verdoheme intermediate. This oxidative heme degradation by HmuS required molecular oxygen and electrons supplied by either ascorbate or NADPH. Electrons could not be directly transferred from NADPH to heme; instead, ferredoxin-NADP+ reductase (FNR) functioned as a mediator. Although HmuS does not share amino acid sequence homology with heme oxygenase (HO), a well-known heme-degrading enzyme, absorption and resonance Raman spectral analyses suggest that the heme iron is coordinated with an axial histidine residue and a water molecule in both enzymes. The substitution of axial His196 or distal Arg102 with an alanine residue in HmuS almost completely eliminated heme-degradation activity, suggesting that Fe-His coordination and interaction of a distal residue with water molecules in the heme pocket are important for this activity.