• Journal of Microbiology and Biotechnology
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• v.16 no.8
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• pp.1325-1329
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• 2006

Structural modeling of $\beta$-galactosidase from L. lactis ssp. lactis 7962 has shown that the residues Cys-472 and His-509 are located in the wall of the active-site cavity. To examine the functions of Cys-472 and His-509, we generated five site-specific mutants: Cys-472-Ser, Cys-472-Thr, Cys-472-Met, His-509-Asn, and His-509-Phe. $\beta$-Galactosidase substituted at Cys-472 with Met or His-509 with Phe had <3% of the activity of the native enzyme when assayed using ONPG as substrate. The other mutants Cys-472-Ser, Cys-472-Thr, and His-509-Asn had ca. 10-15% of the native enzyme activity. The V$_max$ values of the five mutated enzymes were lower (60-7,000-fold) than that of native enzyme. These results show that the catalytic ability of $\beta$-galactosidase is significantly affected by mutations at Cys-472 or His-509.

• BMB Reports
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• v.34 no.5
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• pp.472-477
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• 2001

Ornithine decarboxylase (ODC, EC 4.1.1.17) is the first and key enzyme in eukaryotic polyamine biosynthesis. The cDNA encoding ornithine decarboxylase from Nicotiana glutinosa was cloned ($GeBank^{TM}$ AF 323910) and expressed in E. coli. Site directed mutagenesis were performed on several highly conserved cysteine residues. Among the mutants, C115A showed significant changes in the kinetic properties. The $K_m$ value of the C115A mutant was $1790\;{\mu}M$, which was 3-fold higher than that of the wild-type ODC. There was a dramatic decrease in the $k_{cat}$, values of the C115A mutant, compared to that of the wild-type ODC, which had a $k_{cat}$ value of $77.75\;s^{-1}$. C115A caused a shift in the optimal pH from 8.0 to 8.4. Considering these results, we suggest that cys-115 is involved in the catalytic activity of N. glutinosa ODC.

• Molecules and Cells
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• v.28 no.5
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• pp.463-472
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• 2009

Although the possible cellular roles of several ubiquitin-specific proteases (UBPs) were identified in Arabidopsis, almost nothing is known about UBP homologs in rice, a monocot model plant. In this report, we searched the rice genome database (http://signal.salk.edu/cgi-bin/RiceGE) and identified 21 putative UBP family members (OsUBPs) in the rice genome. These OsUBP genes each contain a ubiquitin carboxyl-terminal hydrolase (UCH) domain with highly conserved Cys and His boxes and were subdivided into 9 groups based on their sequence identities and domain structures. RT-PCR analysis indicated that rice OsUBP genes are expressed at varying degrees in different rice tissues. We isolated a full-length cDNA clone for OsUBP6, which possesses not only a UCH domain, but also an N-terminal ubiquitin motif. Bacterially expressed OsUBP6 was capable of dismantling K48-linked tetra-ubiquitin chains in vitro. Quantitative real-time RT-PCR indicated that OsUBP6 is constitutively expressed in different tissues of rice plants. An in vivo targeting experiment showed that OsUBP6 is predominantly localized to the nucleus in onion epidermal cells. We also examined how knock-out of OsUBP6 affects developmental growth of rice plants. Although homozygous T3 osubp6 T-DNA insertion mutant seedlings displayed slower growth relative to wild type seedlings, mature mutant plants appeared to be normal. These results raise the possibility that loss of OsUBP6 is functionally compensated for by an as-yet unknown OsUBP homolog during later stages of development in rice plants.