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,Elin Moe,Ida Helene Steen,Aurora Martinez,Ingar Leiros,Marte Innselset,Angel L Pey

doi:10.1074/jbc.m610174200

Hanna-Kirsti S Leiros, Elin Moe + Show 5 more

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https://doi.org/10.1074/jbc.m610174200

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Abstract

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 (BH(4)) as cofactor and at 25 degrees C (15 degrees 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)(BH(4))(= 0.3 +/- 0.1 mm), as well as high catalytic efficiency at 10 degrees C. However, the half-inactivation and denaturation temperature is only slightly lowered (T(m) approximately 52 degrees 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 BH(4) (Gly(87)/Phe(88)/Gly(89)) and l-Phe (Tyr(114)-Pro(118)). 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.

Highlights

JULY 27, 2007 VOLUME 282 NUMBER 30 shown by comparative genome analysis [2]
Reverse transcriptase PCR confirmed that phenylalanine hydroxylase (PAH) was expressed in C. psychrerythraea cultivated under these conditions (Fig. 2A)
The fact that C. psychrerythraea lacks an ortholog of homogentisate dioxygenase catalyzing the oxidation of homogentisic acid to 4-maleylacetoacetate, a step needed for the complete oxidation of L-Phe to carbon dioxide and water [65], further supports the hypothesis that PAH is not involved in catabolism but in specialized synthesis of metabolites derived from L-Phe and L-Tyr in C. psychrerythraea

Summary

Introduction

JULY 27, 2007 VOLUME 282 NUMBER 30 shown by comparative genome analysis [2]. To obtain insight into the molecular determinants for cold-active enzymes of this organism, Methe et al [2] prepared 2026 structure models based on 176 templates from the Protein Data Bank. A few enzymes from C. psychrerythraea have been isolated and characterized in detail These included an extracellular cold-active aminopeptidase, which shows characteristic coldadapted activity and structural properties [3] and two isocitrate dehydrogenase isozymes, one found to be monomeric and coldactive, and one found to be dimeric with a mesophilic activity profile [4]. PAH is a non-heme iron-dependent enzyme, which in mammals is found mainly in the liver catalyzing the hydroxylation of L-Phe using molecular oxygen and (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) as co-substrates. In mammals this is the first step in the catabolism of L-Phe to carbon dioxide and water. Bacterial PAH crystal structure determination has been reported only for CvPAH, which shares the fold of the catalytic domain of human and rat PAHs [14]

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