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Deep insights into the history of hexokinases evolution based on mutational pressure theory S.V. Lelevich
Grodno State Medical University, Belarus, Grodno, Gorkogo, 80, slelevich@yandex.by

V.V. Khrustalev, E.V. Barkovsky
Belarussian State Medical University, Belarus, Minsk, Dzerzinskogo, 83, vvkhrustalev@mail.ru, barkovsky@hotmail.ru

There are four genes coding for four types of hexokinases containing two homologous halves in genomes of vertebrates: hexokinase I (HKI); hexokinase II (HKII), hexokinase III (HKIII) and hexokinase domain containing protein I (HKDCI) [1]. It was shown that N-halves of HKI and HKIII are not catalytically active, unlike their C-halves [1]. N-half of HKDCI also possesses mutations which should lead to the loss of its catalytic function [1]. In contrast, both halves of HKII are able to catalyze phosphorylation of hexoses [1]. Nucleotide sequences of coding regions have been downloaded from Ensembl database and from GenBank. N-halves have been separated from C-halves with the help of REPRO algorithm (http://www.ibi.vu.nl/programs/reprowww/). Then they have been aligned by Muscle algorithm included in MEGA 5 program [2]. JTT amino acid evolutionary distances have been calculated between all the N-halves and compared by two-tailed paired t-test with evolutionary distances between all the C-halves for each enzyme. N-half of HKDCI evolves faster than C-half (average ratio between evolutionary distances is equal to 1.147; P = 2.459· 10-15; number of pairwise distances is equal to 171). N-half of HKIII evolves 1.853 times faster than its C-half (P = 4.241· 10-48; N = 120). Although both halves of hexokinase II are catalytically active, evolutionary distances between N-halves of this enzyme are significantly higher (P = 6.437· 10-13; N = 231) than evolutionary distances between its C-halves (average ratio is equal to 1.107). Surprisingly, evolutionary distances between catalytically inactive N-halves of hexokinase I are significantly lower (P = 2.035· 10-10; N = 190) than evolutionary distances between its catalytically active C-halves


(average ratio is equal to 0.886). This fact is in conflict with widely accepted hypothesis stating that N-halves of all hexokinases evolve faster than C-halves [1]. The most of the sequences encoding hexokinases are under the influence of symmetric mutational GC-pressure [3], since GC-content in their third codon positions (3GC) is higher than 50%. A few sequences with 3GC < 50% were excluded from phylogenetic analyses. We calculated average GC-content in first and second codon positions in homologous parts of N- and C-halves for each sequence coding for HKI, HCDCI, HKII and HKIII. Average "(1GC + 2GC) / 2" levels for regions coding for N-halves of HKI, HKDCI and HKIII are higher than those for regions coding for their C-halves (P-values are: 5.752· 10-6; 3.766· 10
-3

and 5.066· 10-11, respectively). The level of "(1GC + 2GC) / 2" is lower in regions coding for N-halves of HKII than in regions coding for C-halves of HKII (P-value is equal to 0.02118). Since average GC-content in first and second codon positions is a kind of a retrospective index, we may suggest that there was a period of time when negative selection was weaker for C-half of HKII than for N-half. Then amino acid sequence of HKII C-half has been stabilized by negative selection and the rates of amino acid substitutions fixation became lower. There had been a period of the "drift" towards higher GC-content in first and second codon positions in the evolutionary history of a region encoding N-half of HKI. Then amino acid sequence of HKI N-half has been stabilized by negative selection, probably, after the acquirement of new functions. 1. D.M. Irwin, H. Tan, (2008) Molecular evolution of the vertebrate hexokinase gene

family: Identification of a conserved fifth vertebrate hexokinase gene, Comp. Biochem. Physiol., Part D 3: 96-107. 2. K. Tamura, D. Peterson, N. Peterson, G. Stecher, M. Nei, S. Kumar, (2011) MEGA5:

Molecular Evolutionary Genetics Analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods, Mol. Biol. Evol., 28: 2731-2739. 3. N. Sueoka (1998) Directional mutation pressure and neutral molecular evolution, Proc.

Natl. Acad. Sci. USA, 85: 2653­2657.