MhuD from Mycobacterium tuberculosis: Probing a Dual Role in Heme Storage and Degradation.

The Mycobacterium tuberculosis (Mtb) heme oxygenase MhuD liberates free iron by degrading heme to the linear tetrapyrrole mycobilin. The MhuD dimer binds up to two hemes within the active site of each monomer. Binding the first solvent-exposed heme allows heme degradation and releases free iron. Binding a second heme renders MhuD inactive, allowing heme storage. Native-mass spectrometry revealed little difference in binding affinity between solvent-exposed and solvent-protected hemes. Hence, diheme-MhuD is formed even when a large proportion of the MhuD population is in the apo form. Apomyoglobin heme transfer assays showed MhuD-diheme dissociation is far slower than monoheme dissociation at ∼0.12 min-1 and ∼0.25 s-1, respectively, indicating that MhuD has a strong affinity for diheme. MhuD has not evolved to preferentially occupy the monoheme form and, through formation of a diheme complex, it functions as part of a larger network to tightly regulate both heme and iron levels in Mtb.

Astaxanthin binding protein in Atlantic salmon.

The rubicund pigmentation in salmon and trout flesh is unique and is due to the deposition of dietary carotenoids, astaxanthin and canthaxanthin in the muscle. The present study was undertaken to determine which protein was responsible for pigment binding. Salmon muscle proteins were solubilized by sequential extractions with non-denaturing, low ionic strength aqueous solutions and segregated as such into six different fractions. Approximately 91% of the salmon myofibrillar proteins were solubilized under non-denaturing conditions using a protocol modified from a method described by Krishnamurthy et al. [Krishnamurthy, G., Chang, H.S., Hultin, H.O., Feng, Y., Srinivasan, S., Kelleher. S.D., 1996. Solubility of chicken breast muscle proteins in solutions of low ionic strength. J. Agric. Food Chem. 44: 408-415.] for the dissolution of avian muscle. To our knowledge, this is the first time this solubilization approach has been applied to the study of molecular interactions in myofibrillar proteins. Astaxanthin binding in each fraction was determined using an in vitro binding assay. In addition, SDS-PAGE and quantitative densitometry were used to separate and determine the relative amounts of each of the proteins in the six fractions. The results showed that alpha-actinin was the only myofibrillar protein correlating significantly (P<0.05) with astaxanthin binding. Alpha-actinin was positively identified using electrophoretic techniques and confirmed by tandem mass spectroscopy. Purified salmon alpha-actinin bound synthetic astaxanthin in a molar ratio of 1.11:1.00. The study was repeated using halibut alpha-actinin, which was found to have a molar binding ratio of astaxanthin to alpha-actinin of 0.893:1. These results suggest that the difference in pigmentation between white fish and Atlantic salmon is not due to binding capacity in the muscle, but rather differences in the metabolism or transport of pigment.

Broad phenotypic variability in a single pedigree with a novel 1410delC mutation in the PST domain of the PAX6 gene.

The PAX6 mutation present in an individual with aniridia was determined and phenotypic features of immediate relatives carrying the same mutation investigated. Mutation analysis revealed a novel single base deletion 1410delC in the PAX6 gene in ten affected individuals. Clinical features ranged from total aniridia to very mild anterior segment findings. Other findings included partial aniridia, iris stromal hypoplasia, keratitis, cataract, glaucoma, optic disc anomalies and foveal hypoplasia. It appears that independent modifying factors may underlie the variability of the different phenotypic features of the PAX6 mutation.