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J. Biol. Chem., Vol. 282, Issue 30, 21973-21986, July 27, 2007

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Structure of Phenylalanine Hydroxylase from Colwellia psychrerythraea 34H, a Monomeric Cold Active Enzyme with Local Flexibility around the Active Site and High Overall Stability^*

Hanna-Kirsti S. Leiros, Angel L. Pey, Marte Innselset, Elin Moe, Ingar Leiros, Ida Helene Steen^¶, and Aurora Martinez¹

From the Norwegian Structural Biology Centre (NorStruct), Department of Chemistry, University of Tromsø, N-9037 Tromsø, the Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009 Bergen, and the ^¶Department of Biology, University of Bergen, P. O. Box 7800, Jahnebakken 5, 5020 Bergen, Norway

The characteristic of cold-adapted enzymes, high catalytic efficiency at low temperatures, is often associated with low thermostability and high flexibility. In this context, we analyzed the catalytic properties and solved the crystal structure of phenylalanine hydroxylase from the psychrophilic bacterium Colwellia psychrerythraea 34H (CpPAH). CpPAH displays highest activity with tetrahydrobiopterin (BH₄) as cofactor and at 25 °C (15 °C above the optimal growth temperature). Although the enzyme is monomeric with a single L-Phe-binding site, the substrate binds cooperatively. In comparison with PAH from mesophilic bacteria and mammalian organisms, CpPAH shows elevated [S_0.5](L-Phe) (= 1.1 ± 0.1 mM) and K_m(BH₄)(= 0.3 ± 0.1 mM), as well as high catalytic efficiency at 10 °C. However, the half-inactivation and denaturation temperature is only slightly lowered (T_m 52 °C; where T_m is half-denaturation temperature), in contrast to other cold-adapted enzymes. The crystal structure shows regions of local flexibility close to the highly solvent accessible binding sites for BH₄ (Gly⁸⁷/Phe⁸⁸/Gly⁸⁹) and L-Phe (Tyr¹¹⁴–Pro¹¹⁸). Normal mode and COREX analysis also detect these and other areas with high flexibility. Greater mobility around the active site and disrupted hydrogen bonding abilities for the cofactor appear to represent cold-adaptive properties that do not markedly affect the thermostability of CpPAH.

Received for publication, October 31, 2006 , and in revised form, May 29, 2007.

The atomic coordinates and structure factors (code 2v27 and 2v28) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by grants from the Research Council of Norway, Helse-Vest, and the Nanoscience Program at the University of Bergen. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1, Figs. S1 and S2, and Refs. 1 and 2.

¹ To whom correspondence should be addressed. Tel.: 47-55586427; Fax: 47-55586360; E-mail: aurora.martinez{at}biomed.uib.no.

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