NADH-regeneration systems for the synthesis of (S)-4-chloro-3-hydroxybutanoate
(ECHB), a novel NADH-dependent carbonyl reductase
(KaCR1), which reduced ethyl 4-chloroacetoacetate (ECAA) to form (S)-ECHB, was
screened and purified from Kluyveromyces aestuarii and a gene encoding KaCR1 was cloned. Glucose dehydrogenase (GDH) and formate dehydrogenase (FDH) were compared as enzymes for NADH
regeneration using Escherichia coli cells coexpressing
each enzyme with KaCR1. E. coli cells coexpressing
GDH produced 45.6 g/l of (S)-ECHB from 50 g/l of ECAA and E. coli cells coexpressing FDH, alternatively, produced only 19.0 g/l.
The low productivity in the case of FDH was suggested to result from the low
activity and instability of FDH.
PMID: 15056898 [PubMed - in process]
Cytochrome c expression and
mitochondrial biogenesis can be invoked by elevated intracellular Ca(2+) in muscle cells. To characterize the potential role
of Ca(2+) as a messenger involved in mitochondrial
biogenesis in muscle, we determined the effects of the Ca(2+) ionophore A-23187 on the expression of nuclear- and mitochondrially encoded genes. Treatment of myotubes with 1 microM A-23187
for 48-96 h increased nuclear-encoded beta-subunit F(1)ATPase
and malate dehydrogenase
(MDH) mRNA levels by 50-100% (P < 0.05) but decreased mRNA levels of
glutamate dehydrogenase (GDH) by 19% (P < 0.05).
mRNA levels of the cytochrome c oxidase
(COX) nuclear-encoded subunits IV, Vb, and VIc were unchanged, whereas the mitochondrially
encoded subunits COX II and COX III were decreased by 30 and 70%, respectively
(P < 0.05). This was paralleled by a 20% decrease (P < 0.05) in COX
activity. These data suggest that cytoplasmic Ca(2+) differentially regulates the mRNA level of nuclear
and mitochondrial genes. The decline in COX II and III mRNA may be mediated by Tfam, because A-23187 modestly reduced Tfam
levels by 48 h. A-23187 induced time-dependent increases in Egr-1 mRNA, along
with the activation of ERK1/2 and AMP-activated protein kinase.
MEK inhibition with PD-98059 attenuated the increase in Egr-1 mRNA. A-23187
also increased Egr-1, serum response factor, and Sp1 protein expression,
transcription factors implicated in mitochondrial biogenesis. Egr-1 overexpression increased nuclear-encoded cytochrome c transcriptional activation by 1.5-fold (P <
0.05) and reduced GDH mRNA by 37% (P < 0.05) but had no effect on MDH or
mRNA. These results indicate that changes in intracellular Ca(2+)
can modify mitochondrial phenotype, in part via the involvement of Egr-1.
PMID: 15075204 [PubMed - in process]