Biochemical and molecular characterization of alpha-D-galactosidase from coffee beans.
Alpha-D-Galactosidase (alpha-Gal; EC 3.2.1.22) is one of three principal enzymes involved in the modification or degradation of plant cell wall galactomannans. In the present paper it is shown that alpha-galactosidase activities in field-grown coffee beans are variable amongst cultivars of the two species investigated (Coffea arabica and C. canephora var. Robusta). Higher activities were found in Arabica cultivars. Using beans from greenhouse-cultivated C. arabica as a model, we showed that alpha-Gal activity was undetectable in the bean perispem tissue, but increased gradually during the endosperm development, to reach a peak at approximately 30 weeks after flowering (WAF) which coincided with the hardening of the endosperm. Alpha-Gal-specific transcripts detected at 22 and 27 WAF accompanied the peak of alpha-Gal activity, but were reduced to be undetectable in mature beans at 30 WAF, while alpha-Gal activity still persisted. Two isoforms were distinguished in 2-DE profiles of crude protein extracts by N-terminal sequencing analysis. Analysis of two-dimensional gel electrophoresis profiles demonstrated that both isoforms accumulated in a linear fashion throughout grain maturation. Alpha-Gal activity was also observed to increase to high levels during in vitro germination of coffee beans suggesting an important function of this enzyme in this process. Alpha-Gal cDNA sequences from Arabica and Robusta were sequenced and their deduced proteins appeared to be very similar, differing by only eight amino acids. Southern-blot analysis suggests that the enzyme was encoded by at least two genes in C. arabica that could explain the existence of the two isoforms identified in 2-DE profiles.
Marraccini
Pierre
P
Nestlé Research Center, 101, avenue Gustave Eiffel, B.P. 9716, 37097 Tours cedex 2, France.
Rogers
W John
WJ
Caillet
Victoria
V
Deshayes
Alain
A
Granato
Dominique
D
Lausanne
Françoise
F
Lechat
Sylvianne
S
Pridmore
David
D
Pétiard
Vincent
V
D000595:Amino Acid Sequence; D015139:Blotting, Southern; D003069:Coffee / Q000201:enzymology* / Q000254:growth & development; D015180:Electrophoresis, Gel, Two-Dimensional; D018525:Germination; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D012639:Seeds / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D000519:alpha-Galactosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D003069:Coffee; D012333:RNA, Messenger; D000519:alpha-Galactosidase