Abstract
The
araBAD
operon encodes three different enzymes required for catabolism of L-arabinose, which is one of the most abundant monosaccharides in nature. L-ribulokinase, encoded by the
araB
gene
,
catalyses conversion of L-ribulose to L-ribulose-5-phosphate, the second step in the catabolic pathway. Unlike other kinases, ribulokinase exhibits diversity in substrate selectivity and catalyses phosphorylation of all four 2-ketopentose sugars with comparable
k
cat
values. To understand ribulokinase recognition and phosphorylation of a diverse set of substrates, we have determined the X-ray structure of ribulokinase from
Bacillus halodurans
bound to L-ribulose and investigated its substrate and ATP co-factor binding properties. The polypeptide chain is folded into two domains, one small and the other large, with a deep cleft in between. By analogy with related sugar kinases, we identified
447
GG
LPQ
K
452
as the ATP binding motif within the smaller domain. L-ribulose binds in the cleft between the two domains
via
hydrogen bonds with the sidechains of highly conserved Trp126, Lys208, Asp274, and Glu329 and the main chain nitrogen of Ala96. The interaction of L-ribulokinase with L-ribulose reveals versatile structural features that help explain recognition of various 2-ketopentose substrates and competitive inhibition by L-erythrulose. Comparison of our structure to that of the structures of other sugar kinases, revealed conformational variations that suggest domain-domain closure movements are responsible for establishing the observed active site environment.