Homocysteine-Thiolactone - Homocysteine in Protein Structure/Function and Human Disease - page 47

3.5.1.2 Bleomycin Hydrolase

Blmh, named for its ability to hydrolyze the anticancer drug bleomycin, is ubiqui-
tously expressed in mammalian tissues, but its expression level is tissue dependent,
as shown in mice [271, 272], rats [273, 274], rabbits [271, 275, 276], and humans
[277], and is also present in other species [278, 279]. Blmh is studied in the context
of Hcy toxicity [85, 251], cancer therapy [277, 280], Alzheimer's disease
[281-284], Huntington's disease [285], keratinization disorders [286], and protein
breakdown [285, 287]. The human genetic polymorphism BLMH-Ile443Val is
associated with an increased risk for Alzheimer's disease [282].
Human and yeast Blmh have almost identical molecular structures, similar to the
20S proteasome, and belong to a family of self-compartmentalizing intracellular
cysteine proteases [278, 288]. Its evolutionary conservation and wide distribution
suggests that Blmh has a conserved cellular function.
Its physiological function was unknown until 2006 when it was shown that Blmh
is a major Hcy-thiolactonase in humans and yeast and that it protects against Hcy
toxicity in yeast [85]. Recombinant human and yeast Blmh proteins, expressed in E.
coli, exhibit Hcy-thiolactonase activities similar to those of native purified
enzymes. Active site mutations, C73A for the human Blmh and H369A for the
yeast Blmh, inactivate the Hcy-thiolactonase activity. Furthermore, yeast Blmh-null
strains are deficient in Hcy-thiolactonase activity, produce more Hcy-thiolactone,
and exhibit greater sensitivity to Hcy toxicity than wild-type yeast. These results
show that Blmh protects cells against Hcy toxicity by hydrolyzing Hcy-thiolactone
[85].
Substrate specificity studies of purified human Blmh show that the enzyme
exhibits absolute stereospecificity for L -Hcy-thiolactone, the preferred natural sub-
strate [85]. Methyl esters of L -Cys and L -Met, but not of other L -amino acids, are
also hydrolyzed. However, D -Hcy-thiolactone, D -Met methyl ester,

γ -thiobutyr-

olactone, and L -homoserine lactone are not hydrolyzed by Blmh [85].
Hcy-thiolactonase activity of Blmh is significantly reduced in brains from
Alzheimer's disease patients compared with unaffected brains [251]. This finding
suggests that diminished functional Blmh activity could contribute to the pathology
of the disease.
Blmh is highly stereoselective and hydrolyzes the L -stereoisomer of Hcy-
thiolactone, while the D -stereoisomer is not hydrolyzed (Fig. 3.2 ) [85]. Comparative
studies of D -Hcy-thiolactone vs. L -Hcy-thiolactone clearances in mice suggest that
Blmh participates in Hcy-thiolactone clearance in vivo [141]. For example, when
wild-type mice are intraperitoneally injected with D -, or D , L -Hcy-thiolactone, 88 %
and 47 % of the injected dose of D - and D , L -Hcy-thiolactone, respectively, is
recovered in mouse plasma (Table 3.11 ). The amounts of recovered D - and D , L -
Hcy-thiolactone are 14- and 7-fold higher, respectively, than the amount L -Hcy-
thiolactone of recovered in mice injected with the L -stereoisomer (6.3 % of the
injected dose). However, half-lives of plasma D -, D , L -, or L -Hcy-thiolactone
are similar (Table 3.11 ). These findings provide evidence that stereoselective