Evolution of New Function in the GTP Cyclohydrolase II Proteins of Streptomyces coelicolor

Abstract

The genome sequence of Streptomyces coelicolor contains three open reading frames (sco1441, sco2687, and sco6655) that encode proteins with significant (>40%) amino acid identity to GTP cyclohydrolase II (GCH II), which catalyzes the committed step in the biosynthesis of riboflavin. The physiological significance of the redundancy of these proteins in S. coelicolor is not known. However, the gene contexts of the three proteins are different, suggesting that they may serve alternate biological niches. Each of the three proteins was overexpressed in Escherichia coli and characterized to determine if their functions are biologically overlapping. As purified, each protein contains 1 molar equiv of zinc/ mol of protein and utilizes guanosine 5′-triphosphate (GTP) as substrate. Two of these proteins (SCO 1441 and SCO 2687) produce the canonical product of GCH II, 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5′-phosphate (APy). Remarkably, however, one of the three proteins (SCO 6655) converts GTP to 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5′-phosphate (FAPy), as shown by UV-visible spectrophotometry, mass spectrometry, and NMR. This activity has been reported for a GTP cyclohydrolase III protein from Methanocaldococcus jannaschii [Graham, D. E., Xu, H., and White, R. H. (2002) Biochemistry 41, 15074-15084], which has no amino acid sequence homology to SCO 6655. Comparison of the sequences of these proteins and mapping onto the structure of the E. coli GCH II protein [Ren, J., Kotaka, M., Lockyer, M., Lamb, H. K., Hawkins, A. R., and Stammers, D. K. (2005) J. Biol. Chem. 280, 36912-36919] allowed identification of a switch residue, Met120, which appears to be responsible for the altered fate of GTP observed with SCO 6655; a Tyr is found in the analogous position of all proteins that have been shown to catalyze the conversion of GTP to APy. The Met120Tyr variant of SCO 6655 acquires the ability to catalyze the conversion of GTP to APy, suggesting a role for Tyr120 in the late phase of the reaction. Our data are consistent with duplication of GCH II in S. coelicolor promoting evolution of a new function. The physiological role(s) of the gene clusters that house GCH II homologues will be discussed.A substantial role for duplication in evolution of new function was proposed (1-3) long before the recent explosion of genome sequencing and annotation projects revealed the pervasiveness of intragenomic duplications in bacterial genomes. Gene duplication is now widely acknowledged to contribute to evolution of new protein functions by releasing the † This research is supported (in part) by a Career Award in the Biomedical Sciences from the Burroughs Wellcome Fund (to V.B.).Additional support for V.B. from the NIH (Grant GM 72623) is gratefully acknowledged. J.E.S. is supported by an NIH predoctoral training grant (GM 08804 GCH II belongs to a class of proteins that catalyze cleavage of carbon-8 of GTP to produce structurally related but distinct products (see Figure 1). GCH I catalyzes the conversio...

Structural and biochemical characterization of GTP cyclohydrolase II from Helicobacter pylori reveals its redox dependent catalytic activity

“…Till now, various biochemical and structural studies have been done to understand the molecular mechanism of GCHII and its role in riboflavin biosynthesis pathway (Fassbinder et al, 2000;Foor and Brown, 1975;Kaiser et al, 2002;Ren et al, 2005;Schramek et al, 2001;Spoonamore et al, 2006). We have characterized GCHII from H. pylori (hGCHII) to design inhibitors against this essential enzyme, which could be developed further as potential anti-bacterial drug.…”

“…GCHII has been studied in many organisms (Bereswill et al, 1998;Herz et al, 2000;Oltmanns et al, 1969;Spoonamore et al, 2006;Zakal'skii et al, 1990) and detailed biochemical and structural characterization has been done from Escherichia coli (E. coli) (Kaiser et al, 2002;Ren et al, 2005;Ritz et al, 2001;Schramek et al, 2001). Crystal structure of GCHII has been reported in mono-functional and bi-functional form from E. coli (eGCHII) (Ren et al, 2005) and Mycobacterium tuberculosis (M. tuberculosis) (mGCHII) (Singh et al, 2013) respectively.…”

Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers

“…The enzyme properties were studied in the bacteria E. coli (35,123,206), B. subtilis (185), Helicobacter pylori (32), and Streptomyces coelicolor (461), the flavinogenic yeast P. guilliermondii (412,418,425), and the model plant Arabidopsis thaliana (174 (206,365). The reaction starts by pyrophosphate release, which appeared to be the rate-limiting step of the overall reaction, after which imidazole ring opening and elimination of formate occur (206,373).…”

“…Archaea and some eubacteria also contain another GTP cyclohydrolase, GTP cyclohydrolase III, which catalyzes the conversion of GTP to 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5-phosphate, i.e., the formylated derivative of the product of GTP cyclohydrolase II (151,181,461). In other words, GTP cyclohydrolase III, in contrast to GTP cyclohydrolase II, hydrolyzes the imidazole ring of GTP but does not remove the resulting formyl group from the formamide.…”

“…Thus, the 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinedione phosphate, the product of GTP cyclohydrolase II, is produced in archaea by the consecutive action of GTP cyclohydrolase III and formamide hydrolase. It is also interesting that the gene coding for GTP cyclohydrolase III from archaea has no homology with genes coding for GTP cyclohydrolase II, whereas the genes display high homology in S. coelicolor (461).…”