PubMed · 9590080
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Y Ogawa, T Hatano. 1998. [Primary hyperoxaluria type 2(PH2)].. https://pubmed.ncbi.nlm.nih.gov/9590080/
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Manganese peroxidase and lignin peroxidase are ligninolytic heme-containing enzymes secreted by the white-rot fungus Phanerochaete chrysosporium. Despite structural similarity, these peroxidases oxidize different substrates. Veratryl alcohol is a typical substrate for lignin peroxidase, while manganese peroxidase oxidizes chelated Mn2+. By a single mutation, S168W, we have added veratryl alcohol oxidase activity to recombinant manganese peroxidase expressed in Escherichia coli. The kcat for veratryl alcohol oxidation was 11 s-1, Km for veratryl alcohol approximately 0.49 mM, and Km for hydrogen peroxide approximately 25 microM at pH 2.3. The Km for veratryl alcohol was higher and Km for hydrogen peroxide was lower for this manganese peroxidase mutant compared to two recombinant lignin peroxidase isoenzymes. The mutant retained full manganese peroxidase activity and the kcat was approximately 2.6 x 10(2) s-1 at pH 4.3. Consistent with relative activities with respect to these substrates, Mn2+ strongly inhibited veratryl alcohol oxidation. The single productive mutation in manganese peroxidase suggested that this surface tryptophan residue (W171) in lignin peroxidase is involved in catalysis.
Microsomal enzymes that catalyze the first step in the biosynthesis of retinoic acid from retinal, retinol dehydrogenases (RDHs), access retinol bound to cellular retinol-binding protein (CRBP). This study tested the hypothesis that the RDHs interact with the region in CRBP designated as the "helical cap" by evaluating single site-directed mutations, namely, L29A, I32E, L35A, L35E, L35R, L36A, F57A, R58A, and R58E. UV analysis showed mutants had similar conformations of retinol in their binding pockets. Nevertheless, the mutants bound retinol with affinities 2-5-fold lower than wild type, except for L35 mutants, which had affinities similar to wild type. All mutants' holoforms had more relaxed conformations about their helical caps, judged by sensitivity to partial protease digestion. Mutants showed no significant differences in Km values, but two (L36A, R58A) had increased Vm values and L35 mutants had decreased Vm values. Overall, the data indicate that the residues tested contribute in varying degrees to CRBP rigidity, retinol binding, and RDH recognition/access to bound retinol. The extent of contributions can be distinguished for several residues. For example, L35 mutants had lower kcat values than wild-type CRBP; thus, L35 seems important for RDH access to retinol. F57, on the other hand, a suspected key residue in controlling retinol entrance/exit, does not make a singular contribution to retinol binding. These results suggest a role for the helical cap region as a locus for RDH interaction and as a portal for ligand access to CRBP, and show that the affinity (Kd) of CRBP for retinol alone does not determine the efficiency of holo-CRBP as substrate. These are the first experimental data of enzyme recognition by a specific exterior residue of CRBP (L35).
This report describes the activity of recombinant human Rdh5 (11-cis-retinol dehydrogenase) with steroids and retinoids and expression of the Rdh5 mRNA in extra-ocular human tissue. The data show that Rdh5 catalyses 9-cis-retinol metabolism equally efficiently as 11-cis-retinol metabolism and recognizes 5alpha-androstan-3alpha,17beta-diol and androsterone as substrates (3alpha-hydroxysteroid dehydrogenase activity), but not testosterone, dihydrotestosterone, oestradiol and corticosterone (lack of 17beta-hydroxysteroid and 11beta-hydroxysteroid dehydrogenase activities). Rdh5 mRNA expression was widespread in extra-ocular tissues with human liver (100% relative expression in extra-ocular tissues only) and mammary gland (97% relative to liver) showing the most intense signals. Other noteworthy relatively intense expression sites included colon (45%), thymus (43%), small intestine (39%), kidney (37%), bladder (29%), pancreas and spleen (28% each), heart (26%), uterus and ovary (25% each), testis (22%) and spinal cord (24%). Human fetal tissues also expressed Rdh5 with fetal liver showing the most intense expression among the fetal tissues (20%). Considered along with the identical nucleotide sequences in the untranslated regions of human Rdh5 and human 9-cis-retinol dehydrogenase cDNAs and the nearly identical nucleotide sequences overall (99% identity), the current results suggest that the two cDNAs represent a single gene product.