SLC24A5 encodes a trans-Golgi network protein with potassium-dependent sodium-calcium exchange activity that regulates human epidermal melanogenesis.

A non-synonymous single nucleotide polymorphism in the human SLC24A5 gene is associated with natural human skin color variation. Multiple sequence alignments predict that this gene encodes a member of the potassium-dependent sodium-calcium exchanger family denoted NCKX5. In cultured human epidermal melanocytes we show using affinity-purified antisera that native human NCKX5 runs as a triplet of approximately 43 kDa on SDS-PAGE and is partially localized to the trans-Golgi network. Removal of the NCKX5 protein through small interfering RNA-mediated knockdown disrupts melanogenesis in human and murine melanocytes, causing a significant reduction in melanin pigment production. Using a heterologous expression system, we confirm for the first time that NCKX5 possesses the predicted exchanger activity. Site-directed mutagenesis of NCKX5 and NCKX2 in this system reveals that the non-synonymous single nucleotide polymorphism in SLC24A5 alters a residue that is important for NCKX5 and NCKX2 activity. We suggest that NCKX5 directly regulates human epidermal melanogenesis and natural skin color through its intracellular potassium-dependent exchanger activity.

Efficient catalytic promiscuity in an enzyme superfamily: an arylsulfatase shows a rate acceleration of 10(13) for phosphate monoester hydrolysis.

We report a second catalytic activity of Pseudomonas aeruginosa arylsulfatase (PAS). Besides hydrolyzing sulfate monoesters, this enzyme catalyzes the hydrolysis of phosphate monoesters with multiple turnovers (>90), a k(cat) value of 0.023 s(-1), a K(M) value of 29 microM, and a kcat/K(M) ratio of 790 M(-1) s(-1) at pH 8.0. This corresponds to a remarkably high rate acceleration of 10(13) relative to the nonenzymatic hydrolysis [(k(cat)/K(M))/k(w)] and a transition-state binding constant (K(tx)) of 3.4 pM. Promiscuous phosphatase and original sulfatase activities only differ by a factor of 620 (measured by k(cat)), so the enzyme provides high accelerations for both reactions. The magnitudes and relative similarity of the kinetic parameters suggest that a functional switch from sulfatase to phosphatase activities is feasible, either by gene duplication or by direct evolution via an intermediate enzyme with dual specificity.

Cloning, expression, and site-directed mutagenesis of the propene monooxygenase genes from Mycobacterium sp. strain M156.

Propene monooxygenase has been cloned from Mycobacterium sp. strain M156, based on hybridization with the amoABCD genes of Rhodococcus corallinus B276. Sequencing indicated that the mycobacterial enzyme is a member of the binuclear nonheme iron monooxygenase family and, in gene order and sequence, is most similar to that from R. corallinus B-276. Attempts were made to express the pmoABCD operon in Escherichia coli and Mycobacterium smegmatis mc(2)155. In the former, there appeared to be a problem resolving overlapping reading frames between pmoA and -B and between pmoC and -D, while in the latter, problems were encountered with plasmid instability when the pmoABCD genes were placed under the control of the hsp60 heat shock promoter in the pNBV1 vector. Fortuitously, constructs with the opposite orientation were constitutively expressed at a level sufficient to allow preliminary mutational analysis. Two PMO active-site residues (A94 and V188) were targeted by site-directed mutagenesis to alter their stereoselectivity. The results suggest that changing the volume occupied by the side chain at V188 leads to a systematic alteration in the stereoselectivity of styrene oxidation, presumably by producing different orientations for substrate binding during catalysis. Changing the volume occupied by the side chain at A94 produced a nonsystematic change in stereoselectivity, which may be attributable to the role of this residue in expansion of the binding site during substrate binding. Neither set of mutations changed the enzyme's specificity for epoxidation.