Enzyme Structure and Dynamics


One Protein, Two Enzymes

Another class of metalloenzymes that we are investigating are from the methionine salv
age pathway (MSP).  The MSP is a ubiquitous biochemical pathway the maintains methionine levels in vivo by recycling the thiomethyl moiety of methionine through a degradation pathway that leads from S-adenosyl methionine (SAM) through methylthioadenosine (MTA) ( Scheme 1). The MSP plays an important (if still not well-understood) role in the cell cycle and carcinogenesis.   The structures of two enzymes in the pathway, the enolase-phosphatase E1 and acireductone dioxygenase (ARD) were determined in our laboratory. Both of these enzymes were discovered in the laboratory of the late Prof. R. Abeles in the Brandeis Biochemistry department.

A particularly interesting feature of ARD is that its functionality changes depending on which metal ion is bound to it (hence the “one-protein two-enzymes” description). ARD to which Ni(II) is bound (NiARD) catalyzes the off-MSP pathway decomposition of acireductone substrate to the (n-2) carboxylic acid, carbon monoxide and formate. From the same substrate, ARD to which Fe(II) is bound generates the on-MSP pathway (n-1) α-keto acid and formate. The function (if any) of CO is unknown, although there are indications that it may play roles in apoptosis signaling and neurotransmission. 

We have determined the solution structure of NiARD (Figure 1) and investigated the roles played by the metal ions in the course of the chemistry catalyzed by ARD. We have also determined the structure of the Fe-containing form ARD (Figure 1)  and found an interesting structural entropy switch that is triggered by the metal ion bound in the active site, and results in extensive changes in secondary structural features of the protein.

Fig. 1. Comparison of the structures of NiARD and FeARD. Letters reference to the ARD sequence as follows: A (Ala 2-Phe 6), B (Leu 15-Ser 18), C (Glu 23-Lys 31), E (Thr 50-Tyr 57), E' (Ile 61-Lys 68), F (Ser 72-Leu 78), G (Lys 85-Glu 90), H (Phe 92-Glu 95), I (Arg 104-Val 107), J (Gly 111-Ile 117), K (Glu 120-Leu 125), L (Asn 129-Ile 132), M (His 140-Met 144), N (Phe 150-Phe 156), O (Gly 161-Gly 168), P (Ile 171-Ala 174). The positions of metal ions are indicated by blue (Ni+2) and gray (Fe+2) spheres. Residues 157-175 (loop O and helix P in NiARD) are disordered in FeARD, and so for clarity are not shown in the FeARD structure.


“Aci-reductone dioxygenase I (ARDI) is an effector of the heterotrimeric G protein beta subunit in Arabidopsis” (E. J. Friedman, H. X. Wang, K. Jiang, I. Perovic, A. Deshpande, T. C. Pochapsky, B. R. S. Temple, S. N. Hicks, T. K. Harden, and A. M. Jones) J. Biol. Chem. 286, 30107-30118 (2011).

"Characterization of metal binding in the active sites of acireductone dioxygenase isoforms from Klebsiella ATCC 8724"  (S. Chai, T. Ju, M. Dang, R. Goldsmith, M. J. Maroney and T. C. PochapskyBiochemistry 47, 2428 -2438 (2008).

"Expression and function of the human androgen-responsive gene ADI1 in prostate cancer" (S. Oram, G. M. Pagani, M. R. Hitchens, J. Stern, S. Eggener, M. Pins, W. Xiao, X. Cai,  R. Haleem, F. Jiang, T. C. Pochapsky, L. Hedstrom and Z. Wang)  Neoplasia 9, 643-651 (2007).

“One protein, two enzymes revisited:  A structural entropy switch interconverts the two isoforms of acireductone dioxygenase” (T. Ju, R. B. Goldsmith, S. C. Chai, M. J. Maroney, S. S. Pochapsky and T. C. Pochapsky)  J. Mol. Biol. 393, 823-834 (2006).

“Nickel in acireductone dioxygenase” (T. C. Pochapsky, T. Ju, B. OuYang, M. Dang, R. Beaulieu, and G.M. Pagani) in "Nickel and Its Surprising Impact in Nature", Vol. 2 of 'Metal Ions in Life Sciences';  A. Sigel, H. Sigel, R. K. O. Sigel, Eds.; John Wiley & Sons, Ltd., Chichester, UK, pp. 473-500 (2007).

" A refined model for the structure of acireductone dioxygenase from Klebsiella ATCC 8724 incorporating residual dipolar couplings”  (T. C. Pochapsky, S. S. Pochapsky , T. Ju,  C. Hoefler and J. Liang) J. Biomol. NMR 34, 117-127 (2006).

"OsARD1 is an immediate-early ethylene response gene involved in recycling of the ethylene precursor S-adenosylmethionine" (M. Sauter, R. Lorbiecke, B. OuYang, T. C. Pochapsky and G. Rzewuski) The Plant Journal 44, 718-729 (2005).

"1H, 13C and 15N chemical shift assignments of an enolase-phosphatase, E1, from Klebsiella oxytoca."  (M. Kostic and T. C. Pochapsky), J. Biomol. NMR 30, 359-360 (2004).

"Analogs of 1-phospho-2,3-dioxo-5-methylthiopentane, an acyclic intermediate in the methionine salvage pathway: a new preparation and characterization of activity with E1 enolase/phosphatase from Klebsiella oxytoca."  (Y. Zhang, M. Heinsen, M. Kostic, G. Pagani, L. Hedstrom, T. Riera, I. Perovic, B. B. Snider and T. C. Pochapsky), Bioorganic and Medicinal Chemistry 12, 3847-3855 (2004).

"Modeling and experiment yields the solution structure of acireductone dioxygenase from Klebsiella" (T. C. Pochapsky, S. S. Pochapsky, T. Ju, H. Mo, F. Al-Mjeni and M. J. Maroney) Nature Struct. Biol. 9, 966-972 (2002).

“XAS Investigation of the Structure and Function of Ni in Acireductone Dioxygenase(F. Al-Mjeni, T. C. Pochapsky, T. Ju, and M. J. Maroney) Biochemistry 41, 6761-6769 (2002).

“Mechanistic studies of two dioxygenases in the methionine salvage pathway of Klebsiella pneumoniae” (Y. Dai, T. C. Pochapsky and R. H. Abeles) Biochemistry 40, 6379-6387 (2001).

1H, 13C and 15N assignments for a carbon monoxide generating metalloenzyme from Klebsiella pneumoniae” (H. Mo, Y. Dai, T. C. Pochapsky and S. S. Pochapsky) J. Biomol. NMR 14, 287-288 (1999).