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Abstract: A membrane-bound pyrroloquinoline quinine (PQQ)-dependent d-sorbitol dehydrogenase (mSLDH) in Gluconobacter oxydans participates in the oxidation of d-sorbitol to l-sorbose by transferring electrons to ubiquinone which links to the respiratory chain. To elucidate the kinetic mechanism, the enzyme purified was subjected to two-substrate steady-state kinetic analysis, product and substrate inhibition studies. These kinetic data indicate that the catalytic reaction follows an ordered Bi Bi mechanism, where the substrates bind to the enzyme in a defined order (first ubiquinone followed by d-sorbitol), while products are released in sequence (first l-sorbose followed by ubiquinol). From these findings, we proposed that the native mSLDH bears two different substrate-binding sites, one for ubiquinone and the other for d-sorbitol, in addition to PQQ-binding and Mg2+-binding sites in the catalytic center. [Copyright &y& Elsevier]

Methylmalonic acidemia (MMA) is caused by the deficient activity of l-methylmalonyl-CoA mutase, which is a vitamin B12 (or cobalamin, Cbl)-dependent enzyme. MMA due to the effect of insufficient Cbl metabolism is classified into three forms (cblA, cblB, and cblH). Recently, the genes responsible for cblA and cblB were identified as MMAA and MMAB, respectively. The MMAA protein likely transports Cbl into the mitochondria for adenosylcobalamin synthesis, while the MMAB protein appears to be an adenosyltransferase. We performed a mutation analysis of 10 unrelated Japanese patients with vitamin B12-responsive MMA. Seven patients had mutations in MMAA, whereas the other three patients showed no disease-causing substitutions in either MMAA or MMAB. Five novel mutations were identified in MMAA (R22X, R145X, L217X, R359G, and 503delC). The 503delC mutation was observed in five of the seven MMAA patients, suggesting that the mutation is prevalent in Japanese patients. This finding may facilitate the DNA diagnosis of vitamin B12-responsive MMA within the Japanese population. [Copyright &y& Elsevier]

Propionic acidemia (PA) is an inborn error of organic acid metabolism caused by a deficiency of propionyl-CoA carboxylase. This enzyme is composed of two non-identical subunits, α and β, which are encoded by the PCCA and PCCB genes, respectively. An enzyme deficiency can result from mutations in either PCCA or PCCB. To elucidate the mutation spectrum in Japanese patients, we have performed a mutation analysis of 30 patients with PA, which included nine previously reported patients. The study revealed that 15 patients were α-subunit deficient and 15 patients were β-subunit deficient. Seven novel mutations were found (IVS18 − 6C > G, 1746G > A, C398R, G197E and IVS18 + 1G > A in the PCCA; A153P and IVS9 + 1G > T in the PCCB). Among these Japanese patients with α-subunit deficiencies, 923–924insT, IVS18 − 6C > G, and R399Q mutations were frequent and the total allelic frequency of these three mutations combined was 56% (17/30). This is in sharp contrast to the mutation spectrum found in Caucasian patients, where no prevalent mutations have been identified. Among the β-subunit deficiencies, there were three frequent mutations; R410W, T428I, and A153P, whose allelic frequencies were 30, 26.7, and 13.3%, respectively. In conclusion, a limited number of mutations are predominant in both PCCA and PCCB genes among Japanese patients with propionic acidemia. [Copyright &y& Elsevier]

Abstract: δ-sarcoglycan, one member of the sarcoglycan complex, is a very conservative muscle-specific protein exclusively expressed in the skeletal and cardiac muscles of vertebrates. Mutations in sarcoglycans are known to be involved in limb-girdle muscular dystrophy (LGMD) and dilated cardiomyopathy (DCM) in humans. To address the role of δ-sarcoglycan gene in zebrafish development, we have studied expression pattern of δ-sarcoglycan in zebrafish embryos and examined the role of δ-sarcoglycan in zebrafish embryonic development by morpholino. Strong expression of δ-sarcoglycan was observed in various muscles including those of the segment, heart, eye, jaw, pectoral fin, branchial arches, and swim bladder in zebrafish embryo. δ-sarcoglycan was also expressed in midbrain and retina. Knockdown of δ-sarcoglycan resulted in severe abnormality in both the cardiac and skeletal muscles. Some severe ones displayed serious morphological abnormality such as hypoplastic head, linear heart, very weak heartbeats, and runtish trunk, all dead within 5dpf. Whole-mount in situ hybridization analysis showed that adaxial cells and muscle pioneers were affected in δ-sarcoglycan knockdown embryos. In addition, absence of δ-sarcoglycan protein severely delayed the cardiac development and influenced the differentiation of cardiac muscle, and the cardiac left-right asymmetry was dramatically changed in morpholino-treated embryos. These data together suggest that δ-sarcoglycan plays an important role in early heart and muscle development. [Copyright &y& Elsevier]

Abstract: Tax-interacting protein-1 (TIP-1) is an unusual signaling protein, containing a single PDZ domain. TIP-1 is able to bind β-catenin with high affinity and thus inhibit its transcriptional activity. The high-resolution crystal structure of TIP-1 in complex with the C-terminal peptide of β-catenin provides molecular details for the recognition of β-catenin by TIP-1. Moreover, structural comparison of peptide-free and peptide-bound TIP-1 reveals that significant conformational changes are required in the βB-βC loop region of TIP-1 to avoid clashes with the incoming C-terminal β-catenin peptide. Such conformational changes have not been observed in other structures of PDZ domains. In addition to the canonical peptide-binding pocket of the PDZ domain, TIP-1 can form a binding cavity to anchor more amino acids through a conserved hydrophobic residue pair (Trp776 of β-catenin and Pro45 of TIP-1). Structural and biochemical data indicate that the canonical binding pocket together with the hydrophobic residue pair are presumably the major cause of the significantly higher affinity of the β-catenin C-terminal to TIP-1 than to other PDZ domains, providing a unique binding specificity. Our results reveal the molecular mechanism of TIP-1 as an antagonist in PDZ domain signaling. [Copyright &y& Elsevier]