| Tahzeeba
Hossain |
's Laboratory |
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Current Research
Folate cofactors are essential in synthesis of purine, pyrimidne,
serine, glycine and methionine. Inadequate intake of folates has been
linked to birth defects of the brain and spinal cord, megaloblastic
anemia, impaired cognitive development, cancer and increased risk of
cardiovascular disease. According to March of Dimes, in the USA each
year 3,000 pregnancies are affected by neural tube defects such as spina
bifida and anencephaly. This number is 10-20 times higher in developing
countries. In humans and animals folate is a dietary requirement because
we are unable to synthesize this vitamin. On the other hand, plants and
certain microbes can synthesize folate de novo. Legumes and green leafy
vegetables are good sources of folates but are not the primary source of
calories in diets for the majority of the population globally. Staple
grains such as rice, wheat, and maize are very low in folate content.
Folate content is also low in tuber and root tuber crops such as potato,
sweetpotato and cassava. Biofortification of staple crops is a cost
efficient way to improve micronutrient deficiency specifically in
developing nations where lack of infrastructure prevents commercial
fortification and distribution of vitamin supplements. Our long term
research goal is to enhance folate contents in cereal and tuber crops.
Our current research focus is biofortification of folate in
tuber crops.
Folate is a tripartite molecule that consists of an aromatic pteridine
ring, a para-aminobenzoic acid (PABA) moiety and an amino acid
glutamate. In bacteria and plants the de novo folate pathway is similar
and consists of two distinct convergent branches. The pterin molecule is
synthesized from guanosine triphosphate (GTP) and PABA from chorismate.
Pterin is coupled with the PABA moiety to form dihydropteroate which is
glutamylated to dihydrofolate and ultimately reduced to form
tetrahydrofolate. In bacteria the pathway is cytosolic. In plants the
pathway is compartmentalized in three subcellular compartments. Pterin
is synthesized in the cytosol and PABA in the plastid. These two
components are combined in mitochondria where the rest of the
tetrahydrofolate synthesis takes place.
We engineered the first step of the pteridine branch of the pathway in
the model plant Arabidopsis thaliana by introducing an E.
coli enzyme from the bacterial de novo folate pathway. The E.
coli enzyme GTP-cyclohydrolase-1 (EcGCH-1) catalyzes the first
committed step of the pathway and is not subjected to feedback
inhibition. Our hypothesis was that expression of E. coli GCH-1
in plants would increase pteridine synthesis which will ultimately lead
to increase in total folate. We observed 1250 fold increase of the
pteridine level in our homozygous transgenic Arabidopsis
expressing E. coli GCH-1 (Fig 1). In these plants the total
folate level only increased two to four fold indicating other rate
limiting steps in the pathway. Total PABA analysis of the homozygous
Arabidopsis plants expressing EcGCH-1 gene indicated severe
depletion of PABA pools. To establish if the PABA supply was limiting we
applied exogenous PABA to homozygous Arabidopsis and measured
total folate levels. In these plants total folate levels increased 8 and
20 fold in leaves and seeds respectively. This suggested availability of
PABA as another rate limiting factor of the de novo folate pathway
leading to engineering of the PABA branch.
PABA synthesis from chorismate
takes place in two steps by three different enzymes in E. coli
whereas in plants it requires two enzymes. In E. coli
chorismate is converted to 4-amino-4-deoxy chorismate (ADC) by PabA and
PabB proteins. In plants chorismate is converted to ADC by ADC synthase,
a single protein with fused domains homologous to bacterial PabA and
PabB. In both E. coli and plants the formation of PABA requires
elimination of pyruvate and aromatization of ADC by ADC lyase. Current
research in our laboratory is focusing on expression of both bacterial
and plant genes side by side to find the best suitable method for
engineering the PABA branch in plants.
Figure 1: HPLC Analysis of Pteridines in
Leaves of Transgenic and Non-transgenic Arabidopsis thaliana.
Pteridine levels increased 1250-fold in transgenic Arabidopsis
(line 2-19) expressing EcGCH-1 over wild type (WT) control. |
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