Journal of Molecular Evolution

, Volume 70, Issue 6, pp 545–556

Evolutionary Bases of Carbohydrate Recognition and Substrate Discrimination in the ROK Protein Family


DOI: 10.1007/s00239-010-9351-1

Cite this article as:
Conejo, M.S., Thompson, S.M. & Miller, B.G. J Mol Evol (2010) 70: 545. doi:10.1007/s00239-010-9351-1


The ROK (repressor, open reading frame, kinase) protein family (Pfam 00480) is a large collection of bacterial polypeptides that includes sugar kinases, carbohydrate responsive transcriptional repressors, and many functionally uncharacterized gene products. ROK family sugar kinases phosphorylate a range of structurally distinct hexoses including the key carbon source d-glucose, various glucose epimers, and several acetylated hexosamines. The primary sequence elements responsible for carbohydrate recognition within different functional categories of ROK polypeptides are largely unknown due to a limited structural characterization of this protein family. In order to identify the structural bases for substrate discrimination in individual ROK proteins, and to better understand the evolutionary processes that led to the divergent evolution of function in this family, we constructed an inclusive alignment of 227 representative ROK polypeptides. Phylogenetic analyses and ancestral sequence reconstructions of the resulting tree reveal a discrete collection of active site residues that dictate substrate specificity. The results also suggest a series of mutational events within the carbohydrate-binding sites of ROK proteins that facilitated the expansion of substrate specificity within this family. This study provides new insight into the evolutionary relationship of ROK glucokinases and non-ROK glucokinases (Pfam 02685), revealing the primary sequence elements shared between these two protein families, which diverged from a common ancestor in ancient times.


ROKSugar kinaseRepressorSubstrate specificityEnzyme evolution

Supplementary material

239_2010_9351_MOESM1_ESM.fsa (176 kb)
Supplementary Figure 1: FASTA formatted alignment of the complete ROK data set containing 227 members. (FSA 175 kb)
239_2010_9351_MOESM2_ESM.fsa (85 kb)
Supplementary Figure 2: FASTA formatted alignment of the complete ROK data set masked at 5%. (FSA 85.3 kb)
239_2010_9351_MOESM3_ESM.fsa (26 kb)
Supplementary Figure 3: FASTA formatted alignment of the complete, merged ROK and non-ROK data set. (FSA 26.3 kb)
239_2010_9351_MOESM4_ESM.tree (14 kb)
Supplementary Figure 4: Phylogenetic tree of the complete ROK data set. (TREE 13.8 kb)
239_2010_9351_MOESM5_ESM.tree (3 kb)
Supplementary Figure 5: Phylogenetic tree of the merged ROK and non-ROK data set. (TREE 3.40 kb)
239_2010_9351_MOESM6_ESM.fsa (333 kb)
Supplementary Figure 6: FASTA formatted ancestral node sequences resulting from phylogenetic analysis of the complete ROK data set. (FSA 333 kb)

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Department of Chemistry and BiochemistryFlorida State UniversityTallahasseeUSA
  2. 2.Department of BiologyValdosta State UniversityValdostaUSA