Structure and Characterization of Phosphoglucomutase 5 from Atlantic and Baltic Herring—An Inactive Enzyme with Intact Substrate Binding

biomolecules Article Structure and Characterization of Phosphoglucomutase 5 from Atlantic and Baltic Herring—An Inactive Enzyme with Intact Substrate Binding 1, 1, , 2 2 Robert Gustafsson y , Ulrich Eckhard y z , Weihua Ye , Erik D. Enbody , Mats Pettersson 2, Per Jemth 2, Leif Andersson 2,3,4 and Maria Selmer 1,* 1 Department of Cell and Molecular Biology, Uppsala University, BMC, Box 596, SE-751 24 Uppsala, Sweden; [email protected] (R.G.); [email protected] (U.E.) 2 Department of Medical Biochemistry and Microbiology, Uppsala University, BMC, Box 582, SE-751 23 Uppsala, Sweden; [email protected] (W.Y.); [email protected] (E.D.E.); [email protected] (M.P.); [email protected] (P.J.); [email protected] (L.A.) 3 Department of Veterinary Integrative Biosciences, Texas A & M University, College Station, TX 77843, USA 4 Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, SE-75007 Uppsala, Sweden * Correspondence: [email protected] These authors contributed equally. y Current address: Proteolysis Lab, Department of Structural Biology, Molecular Biology Institute of z Barcelona, CSIC, Barcelona Science Park, Baldiri Reixac, 15–21, 08028 Barcelona, Catalonia, Spain. Received: 8 November 2020; Accepted: 30 November 2020; Published: 3 December 2020 Abstract: Phosphoglucomutase 5 (PGM5) in humans is known as a structural muscle protein without enzymatic activity, but detailed understanding of its function is lacking. PGM5 belongs to the alpha-D-phosphohexomutase family and is closely related to the enzymatically active metabolic enzyme PGM1. In the Atlantic herring, Clupea harengus, PGM5 is one of the genes strongly associated with ecological adaptation to the brackish Baltic Sea. We here present the first crystal structures of PGM5, from the Atlantic and Baltic herring, differing by a single substitution Ala330Val. The structure of PGM5 is overall highly similar to structures of PGM1. The structure of the Baltic herring PGM5 in complex with the substrate glucose-1-phosphate shows conserved substrate binding and active site compared to human PGM1, but both PGM5 variants lack phosphoglucomutase activity under the tested conditions. Structure comparison and sequence analysis of PGM5 and PGM1 from fish and mammals suggest that the lacking enzymatic activity of PGM5 is related to differences in active-site loops that are important for flipping of the reaction intermediate. The Ala330Val substitution does not alter structure or biophysical properties of PGM5 but, due to its surface-exposed location, could affect interactions with protein-binding partners. Keywords: phosphoglucomutase 5; herring; adaptation; enzyme structure; phosphohexomutase 1. Introduction The Baltic Sea is a unique environment. The salinity of the brackish waters gradually drops to about 2–3% in the Bothnian Bay compared to 35% in the Atlantic Ocean [1]. Herring is one of few marine fishes that reproduce throughout the Baltic Sea. Already Linneaus divided the Atlantic and Baltic herring into two separate subspecies, Clupea harengus harengus and Clupea harengus membras [2], but early genetic studies based on a limited number of markers were unable to separate them genetically [3]. Thus, it was thought that the difference in appearance between the small and lean Baltic herring and the larger and fatter Atlantic herring was purely from environmental origin. More recent studies Biomolecules 2020, 10, 1631; doi:10.3390/biom10121631 www.mdpi.com/journal/biomolecules Biomolecules 2020, 10, x FOR PEER REVIEW 2 of 24 Biomolecules 2020, 10, 1631 2 of 23 genetically [3]. Thus, it was thought that the difference in appearance between the small and lean Baltic herring and the larger and fatter Atlantic herring was purely from environmental origin. More recentbased onstudies whole based genome on whole sequencing genome of sequencing population of samples population have samples completely have changedcompletely the changed picture theand picture revealed and hundreds revealed of hundreds loci showing of loci strong showing genetic strong diff geneticerentiation differentiation between the between Atlantic the and Atlantic Baltic andherring Baltic [1, herring4,5]. This [1,4,5] ecological. This ecological adaptation adaptation must be recent, must be as recent, the Baltic as the Sea Baltic has only Sea existedhas only since existed the sincelast glaciation the last glaciation about 10,000 about years 10,000 ago years [6]. The ago allele [6]. The frequencies allele frequencies at several at of several these lociof these show loci a strong show acorrelation strong correlation with salinity with at salinity the spawning at the spawning site where site the where fish were the collected.fish were Ancollected. important An important discovery discoveryin these studies in these was studies that missense was that mutations missense have mutations played have a prominent played a role prominent for the adaptation role for the of adaptationherring to the of brackishherring Balticto the Sea brackish [1]. For Baltic instance, Sea it[1] was. For recently instance, reported it was that recently a missense reported mutation that ina missenserhodopsin mutation (Phe261Tyr) in rhodopsin has been critical(Phe261Tyr) for the has adaptation been critical to the for red-shifted the adaptation light environment to the red-shifted in the lightBaltic environment Sea [7]. Here, in wethe exploreBaltic Sea the [7] functional. Here, we significance explore the of functional Ala330Val significance in phosphoglucomutase of Ala330Val in 5 (PGM5),phosphoglucomutase another missense 5 (PGM5), mutation another that shows missense a striking mutation allele that frequency shows a di strikingfference allele (>80%) frequency between differencethe Atlantic (> and80%) Baltic between herring. the Atlantic and Baltic herring. PGM5 belongs to the alpha-D-phosphohexomutasealpha-D-phosphohexomutase family family that most notably includes PGM1, PGM2, PGM2L1,PGM2L1, PGM3,PGM3,and and PGM5 PGM5 [ 8[8–11–11]]. PGM5. PGM5 is is most most closely closely related related to PGM1to PGM1 [8– 10[8–],10] which, which is the is themost most studied studied PGM P andGM theand main the isomerasemain isomerase responsible responsible for catalyzing for catalyzing the interconversion the interconversion between betweenglucose-1-phosphate glucose-1-phosphate (G1P) and (G1P) glucose-6-phosphate and glucose-6- (G6P)phosphate (Figure (G6P)1)[ 9 ].(Figure PGM1 is1) thus[9]. responsiblePGM1 is thus for responsiblethe metabolic for shift the betweenmetabolic glycolysis shift between (which glycolysis uses G6P) (which and gluconeogenesisuses G6P) and gluconeogenesis (which uses G1P) (which [12]. Thisuses G1P) reaction [12] requires. This reaction a phosphorylated requires a phosphorylated serine residue serine that donates residueits that phosphoryl donates its group phosphoryl to the groupsubstrate to the to formsubstrate the intermediate to form the intermediate glucose-1,6-bisphosphate glucose-1,6-bisphosphate (G16P), which (G16P), has to which flip 180 has degrees to flip 180to allow degrees transfer to allow of the transfer other phosphorylof the other group phosphoryl to the enzymegroup to to the regenerate enzyme theto phosphoserineregenerate the (Figurephosphoserine1)[ 9]. The (Figure phosphoryl 1) [9]. The transfer phosphoryl reaction transfer is reversible reaction and is reversible is believed and to occuris believed using to general occur usingacid-base general catalysis acid- [base13]. Basedcatalysis on mutagenesis[13]. Based on studies mutagenesis in bacteria studies [14– 16in ],bacteria a lysine [14 in– a16] loop, a lysine of domain in a loopIII was of domain proposed III aswas catalytic proposed base as catalytic and an argininebase and inan a arginine loop from in a domainloop from I as domain catalytic I as acid catalytic [12]. acidHowever, [12]. However, based on quantumbased on mechanicalquantum mechanical calculations, calculations, an alternative an alternative catalytic acid catalytic histidine acid following histidine followingthe phosphoserine the phosphoserine in loop 1 was in recentlyloop 1 was proposed, recently suggesting proposed, that suggesting the arginine that instead the arginine plays ainstead role in playselectrostatic a role in stabilization electrostatic [17 stabilization]. [17]. Figure 1. GeneralGeneral reaction reaction mechanism of phosphoglucomutase (PGM)(PGM) enzymes.enzymes. P = phosphatephosphate group group.. There is until now no structure available of PGM5, but several crystal structures of apo and ligandligand-bound-bound PGM1PGM1 from from di ffdifferenterent organisms, organisms, including including rabbit rabbit and humanand hum [10an,12 ,[10,12,18,19]18,19]. These. PGM1These PGM1structures structures are well-conserved are well-conserved and are composed and are composed of four domains of four (I–IV) domains that form (I–IV) a bi-lobed that form heart-shaped a bi-lobed heartstructure-shaped with struct a deepure andwith widea deep active-site and wide cleft active between-site cleft the between first two the and first the two last and two the domains. last two domains.Upon ligand Upon binding, ligand domainbinding, IVdomain closes IV the closes active the site active by a site hinge-like by a hinge movement-like movement creating creating a closed a closedconformation conformation [20–22]. [20 One–22] loop. One from loop each from of the each four of domains, the four named domains, 1–4 as named the corresponding 1–4

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