High-level production of recombinant fungal endo-beta-1,4-xylanase in the methylotrophic yeast Pichia pastoris

Efficient production of recombinant Aspergillus niger family 11 1, 4-beta-xylanase was achieved in Pichia pastoris. The cDNA-encoding XylA fused to the Saccharomyces cerevisiae invertase signal peptide was placed under the control of the P. pastoris AOX1 promoter. Secretion yields up to 60 mg/liter were obtained in synthetic medium. The recombinant XylA was purified to homogeneity using a one-step purification protocol and found to be identical to the enzyme overexpressed in A. niger with respect to size, pI, and immunoreactivity. N-terminal sequence analysis of the recombinant protein indicated that the S. cerevisiae signal peptide was correctly processed in P. pastoris. The purified protein has a molecular weight of 19,893 Da, in excellent agreement with the calculated mass, and appears as one single band on isoelectric focusing with pI value around 3.5. Electrospray ionization mass spectrometry confirmed the presence of one major isoform produced by P. pastoris and the absence of glycosylation. The recombinant enzyme was further characterized in terms of specific activity, pH profile, kinetic parameters, and thermostability toward birchwood xylan as substrate and compared with the xylanase purified from A. niger. Both enzymes exhibit a pH optimum at 3.5 and maximal activity at 50 degrees C. The enzyme activity follows normal Michaelis-Menten kinetics with K(m) and V(max) values similar for both enzymes. P. pastoris produced recombinant xylanase in high yields that can be obtained readily as a single form. A. niger xylanase is the first microbial xylanase efficiently secreted and correctly processed by P. pastoris.     

Studies into factors contributing to substrate specificity of membrane-bound 3-ketoacyl-CoA synthases.

We are interested in constructing a model for the substrate-binding site of fatty acid elongase-1 3-ketoacyl CoA synthase (FAE1 KCS), the enzyme responsible for production of very long chain fatty acids of plant seed oils. Arabidopsis thaliana and Brassica napus FAE1 KCS enzymes are highly homologous but the seed oil content of these plants suggests that their substrate specificities differ with respect to acyl chain length. We used in vivo and in vitro assays of Saccharomyces cerevisiae-expressed FAE1 KCSs to demonstrate that the B. napus FAE1 KCS enzyme favors longer chain acyl substrates than the A. thaliana enzyme. Domains/residues responsible for substrate specificity were investigated by determining catalytic activity and substrate specificity of chimeric enzymes of A. thaliana and B. napus FAE1 KCS. The N-terminal region, excluding the transmembrane domain, was shown to be involved in substrate specificity. One chimeric enzyme that included A. thaliana sequence from the N terminus to residue 114 and B. napus sequence from residue 115 to the C terminus had substrate specificity similar to that of A. thaliana FAE1 KCS. However, a K92R substitution in this chimeric enzyme changed the specificity to that of the B. napus enzyme without loss of catalytic activity. Thus, this study was successful in identifying a domain involved in determining substrate specificity in FAE1 KCS and in engineering an enzyme with novel activity.     

The human CLN2 protein/tripeptidyl-peptidase I is a serine protease that autoactivates at acidic pH

The CLN2 gene mutated in the fatal hereditary neurodegenerative disease late infantile neuronal ceroid lipofuscinosis encodes a lysosomal protease with tripeptidyl-peptidase I activity. To understand the enzymological properties of the protein, we purified and characterized C-terminal hexahistidine-tagged human CLN2p/tripeptidyl-peptidase I produced from insect cells transfected with a baculovirus vector. The N terminus of the secreted 66-kDa protein corresponds to residue 20 of the primary CLN2 gene translation product, indicating removal of a 19-residue signal peptide. The purified protein is enzymatically inactive; however, upon acidification, it is proteolytically processed and concomitantly acquires enzymatic activity. The N terminus of the final 46-kDa processed form (Leu196) corresponds to that of mature CLN2p/tripeptidyl-peptidase I purified from human brain. The activity of the mature enzyme is irreversibly inhibited by the serine esterase inhibitor diisopropyl fluorophosphate, which specifically and stoichiometrically reacts with CLN2p/tripeptidyl-peptidase I at Ser475, demonstrating that this residue represents the active site nucleophile. Expression of wild type and mutant proteins in CHO cells indicate that Ser475, Asp360, Asp517, but not His236 are essential for activity. These data indicate that the CLN2 gene product is synthesized as an inactive proenzyme that is autocatalytically converted to an active serine protease.     

Role of proline residues in the folding of serine hydroxymethyltransferase

Previous studies on the folding mechanism of Escherichia coli serine hydroxymethyltransferase (SHMT) showed that the final rate determining folding step was from an intermediate that contained two fully folded domains with N-terminal segments of approximately 55 residues and interdomain segments of approximately 50 residues that were still solvent exposed and subject to proteolysis. The interdomain segment contains 3 Pro residues near its N terminus and 2 Pro residues near its C terminus. The 5 Pro residues were each mutated to both a Gly and Ala residue, and each mutant SHMT was purified and characterized with respect to kinetic properties, stability, secondary structure, and folding mechanism. The results showed that Pro214 and Pro218 near the N terminus of the interdomain segment are not critical for folding, stability, or activity. The P216A mutant also retained most of the characteristics of the native enzyme, but its folding rate was altered. However, the P216G mutant was severely compromised in folding into a catalytically competent enzyme. Mutation of both Pro258 and Pro264 had altered folding kinetics and resulted in enzymes that expressed little catalytic activity. The Phe257-Pro258 bond is cis in its configuration, and the P258A mutant SHMT showed reduced thermal stability. Pro216, Pro258, and Pro264 are conserved in all 53 known sequences of this enzyme. The results are discussed in terms of the role of each Pro residue in maintaining the structure and function of SHMT and a possible role in pyridoxal 5'-phosphate addition to the apo-enzyme.     

Expression of eukaryotic glycosyltransferases in the yeast Pichia pastoris

The methylotrophic yeast Pichia pastoris is often used as an organism for the heterologous expression of proteins and has been used already for production of a number of glycosyltransferases involved in the biosynthesis of N- and O-linked oligosaccharides. In our recent studies, we have examined the expression in P. pastoris of Arabidopsis thaliana and Drosophila melanogaster core alpha1,3-fucosyltransferases (EC 2.4.1.214), A. thaliana beta1,2-xylosyltransferase (EC 2.4.2.38), bovine beta1,4-galactosyltransferase I (EC 2.4.1.38), D. melanogaster peptide O-xylosyltransferase (EC 2.4.2.26), D. melanogaster and Caenorhabditis elegans beta1,4-galactosyltransferase VII (SQV-3; EC 2.4.1.133) and tomato Lewis-type alpha1,4-fucosyltransferase (EC 2.4.1.65). Temperature, cell density and medium formulation have varying effects on the amount of activity resulting from expression under the control of either the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) or inducible alcohol oxidase (AOX1) promoters. In the case of the A. thaliana xylosyltransferase these effects were most pronounced, since constitutive expression at 16 degrees C resulted in 30-times more activity than inducible expression at 30 degrees C. Also, the exact nature of the constructs had an effect; whereas soluble forms of the A. thaliana xylosyltransferase and fucosyltransferase were active with N-terminal pentahistidine tags (in the former case facilitating purification of the recombinant protein to homogeneity), a C-terminally tagged form of the A. thaliana fucosyltransferase was inactive. In the case of D. melanogaster beta1,4-galactosyltransferase VII, expression with a yeast secretion signal yielded no detectable activity; however, when a full-length form of the enzyme was introduced into P. pastoris, an active secreted form of the protein was produced.     

Molecular cloning and characterization of a plant alpha1,3/4-fucosidase based on sequence tags from almond fucosidase I

Our work with almond peptide N-glycosidase A made us interested also in the alpha1,3/4-fucosidase which is used as a specific reagent for glycoconjugate analysis. The enzyme was purified to presumed homogeneity by a series of chromatographic steps including dye affinity and fast-performance anion exchange chromatography. The 63 kDa band was analyzed by tandem mass spectrometry which yielded several partial sequences. A homology search retrieved the hypothetical protein Q8GW72 from Arabidopsis thaliana. This protein has recently been described as being specific for alpha1,2-linkages. However, cDNA cloning and expression in Pichia pastoris of the A. thaliana fucosidase showed that it hydrolyzed fucose in 3- and 4-linkage to GlcNAc in Lewis determinants whereas neither 2-linked fucose nor fucose in 3-linkage to the innermost GlcNAc residue were attacked. This first cloning of a plant alpha1,3/4-fucosidase also confirmed the identity of the purified almond enzyme and thus settles the notorious uncertainty about its molecular mass. The alpha1,3/4-fucosidase from Arabidopsis exhibited striking sequence similarity with an enzyme of similar substrate specificity from Streptomyces sp. (Q9Z4I9) and with putative proteins from rice.     

Expression of glycosylated haloalkane dehalogenase LinB in Pichia pastoris

Heterologous expression of the bacterial enzyme haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26 in methylotrophic yeast Pichia pastoris is reported. The haloalkane dehalogenase gene linB was subcloned into the pPICZalphaA vector and integrated into the genome of P. pastoris. The recombinant LinB secreted from the yeast was purified to homogeneity and biochemically characterized. The deglycosylation experiment and mass spectrometry measurements showed that the recombinant LinB expressed in P. pastoris is glycosylated with a 2.8 kDa size of high mannose core. The specific activity of the glycosylated LinB was 15.6 +/- 3.7 micromol/min/mg of protein with 1,2-dibromoethane and 1.86 +/- 0.36 micromol/min/mg of protein with 1-chlorobutane. Activity and solution structure of the protein produced in P. pastoris is comparable with that of recombinant LinB expressed in Escherichia coli. The melting temperature determined by the circular dichroism (41.7+/-0.3 degrees C for LinB expressed in P. pastoris and 41.8 +/- 0.3 degrees C expressed in E. coli) and thermal stability measured by specific activity to 1-chlorobutane were also similar for two enzymes. Our results show that LinB can be extracellularly expressed in eukaryotic cell and glycosylation had no effect on activity, protein fold and thermal stability of LinB.     

Recombinant expression of molybdenum reductase fragments of plant nitrate reductase at high levels in Pichia pastoris

Mo reductase (MoR; formerly cytochrome c reductase) fragments of NADH:NO(3) reductase (NR; EC1.6.6.1) were cytosolically expressed in Pichia pastoris, a methylotrophic yeast, using spinach (Spinacia oleracea) and corn (Zea maize) cDNAs. In fermenter cultures, spinach MoR was expressed at 420 mg L(-1), corn MoR at 32 mg L(-1), and corn MoR plus with putative NR interface domain N terminus (MoR+) at 17 mg L(-1). Constitutively expressed MoR+ was structurally stable while it was degraded when expressed by methanol induction, which suggests methanol growth produces more proteinase. Methanol-induced expression yielded more target protein. All three MoR were purified to homogeneity and their polypeptides were approximately 41 (MoR) and approximately 66 (MoR+) kD. MoR was monomeric and MoR+ dimeric, confirming the predicted role for dimer interface domain of NR. MoR+, although differing in quaternary structure from MoR, has similar kinetic properties for ferricyanide and cytochrome c reductase activities and visible spectra, which were like NR. Redox potentials of MoR and MoR+ were similar for flavin, whereas MoR+ had a more negative potential for heme-iron. Reaction schemes for MoR catalyzed reactions were proposed based on fast-reaction rapid-scan stopped-flow kinetic analysis of MoR. P. pastoris is an excellent system for producing the large amounts of NR fragments needed for detailed biochemical studies.     

Identification of molecular determinants that are important in the assembly of N-methyl-D-aspartate receptors

To determine which domains of the N-methyl-d-aspartate (NMDA) receptor are important for the assembly of functional receptors, a number of N- and C-terminal truncations of the NR1a subunit have been produced. Truncations containing a complete ligand binding domain bound glycine antagonist and gave binding constants similar to those of the native subunit, suggesting they were folding to form antagonist binding sites. Since NR2A is not transported to the cell surface unless it is associated with NR1 (McIlhinney, R. A. J., Le Bourdellès, B., Tricuad, N., Molnar, E., Streit, P., and Whiting, P. J. (1998) Neuropharmacology 37, 1355-1367), surface expression of NR2A can be used to monitor the association of the subunits. There was progressive loss of NR2A cell surface expression as the N terminus of NR1a was shortened, with complete loss when truncated beyond residue 380. Removal of the C terminus and/or the last transmembrane domain did not affect NR2A surface expression. Similar results were obtained in co-immunoprecipitation experiments. The oligomerization status of the co-expressed NR1a constructs and NR2A subunits was investigated using a non-denaturing gel electrophoresis system (blue native-polyacrylamide gel electrophoresis) and sucrose density gradient centrifugation. The blue native-polyacrylamide gel electrophoresis system also showed that the NR1a subunits could form a homodimer, which was confirmed using soluble constructs of the NR1a subunit. Together these results suggest the residues N-terminal of residue 380 are important for the association of NR2A with NR1a and that the complete N-terminal domain of the NR1a subunit is required for oligomerization with NR2A.     

Mutagenesis of key amino acids alters activity of a Saccharomyces cerevisiae endo-polygalacturonase expressed in Pichia pastoris

A polygalacturonase (PG)-encoding gene from Saccharomyces cerevisiae (PGU1) was successfully expressed in the methylotrophic yeast Pichia pastoris. PG secretion was efficiently directed by the S. cerevisiae alpha-factor signal sequence, while the native (PGU1) leader peptide was unable to direct protein export in P. pastoris. The level of PGU1 activity achieved in P. pastoris was significantly enhanced when compared to activity using the same gene in S. cerevisiae. Expression of PG proteins, engineered by site-directed mutagenesis, in P. pastoris showed that aspartic acid residues at positions 179, 200, and 201, and histidine 222 were essential for enzyme activity. Mutation of the two potential glycosylation sites in PGU1 showed that the two residues individually (N318D, N330D) did not affect secreted enzyme activity, but the double mutant caused a 50% reduction in enzyme activity when compared to the wild-type PGU1 transformant.     

Cloning, expression, and characterization of an oligoxyloglucan reducing end-specific xyloglucanobiohydrolase from Aspergillus nidulans

An oligoxyloglucan reducing end-specific xyloglucanobiohydrolase from the filamentous fungus Aspergillus nidulans was cloned and expressed in Pichia pastoris as a secreted histidine-tagged protein and purified by affinity chromatography. The enzyme acts on xyloglucan oligomers and releases the first two glycosyl residue segments from the reducing end, provided that neither the first glucose nor the xylose attached to the third glucose residue from the reducing end is not further substituted. The enzyme has a specific activity of 7 U/mg at the pH optimum of 3 and at the temperature optimum of 42 degrees C.     

Purification and characterization of keratinase from recombinant Pichia and Bacillus strains

The keratinase gene from Bacillus licheniformis MKU3 was cloned and successfully expressed in Bacillus megaterium MS941 as well as in Pichia pastoris X33. Compared with parent strain, the recombinant B. megaterium produced 3-fold increased level of keratinase while the recombinant P. pastoris strain had produced 2.9-fold increased level of keratinase. The keratinases from recombinant P. pastoris (pPZK3) and B. megaterium MS941 (pWAK3) were purified to 67.7- and 85.1-folds, respectively, through affinity chromatography. The purified keratinases had the specific activity of 365.7 and 1277.7 U/mg, respectively. Recombinant keratinase from B. megaterium was a monomeric protein with an apparent molecular mass of 30 kDa which was appropriately glycosylated in P. pastoris to have a molecular mass of 39 kDa. The keratinases from both recombinant strains had similar properties such as temperature and pH optimum for activity, and sensitivity to various metal ions, additives and inhibitors. There was considerable enzyme stability due to its glycosylation in yeast system. At pH 11 the glycosylated keratinase retained 95% of activity and 75% of its activity at 80 degrees C. The purified keratinase hydrolyzed a broad range of substrates and displayed effective degradation of keratin substrates. The K(m) and V(max) of the keratinase for the substrate N-succinyl-Ala-Ala-Pro-Phe-pNA was found to be 0.201 mM and 61.09 U/s, respectively. Stability in the presence of detergents, surfactants, metal ions and solvents make this keratinase suitable for industrial processes.     

Inactivation of Sunflower NADH:Nitrate Reductase by White Light-Activated Rose Bengal

Chemical modification of purified nitrate reductase (NR) from sunflower leaves by white light-irradiated rose bengal was studied. NADH:NR activity was inhibited by light-activated rose bengal in both a concentration- and time-dependent manner. MV:NR activity was less sensitive to inhibition than NADH:NR activity, especially when the enzyme was preincubated with NADH. Preincubation of the enzyme with FAD protected inhibition of NADH:NR activity but not the MV:NR activity. These results suggest that sunflower NR contains sensitive histidine residue which interacts with reduced FAD during catalytic electron transfer. Most importantly, NADH-reduced NR was more sensitive to the irradiated dye, indicating that conformation of the oxidized and reduced enzyme forms were different.     

Secretion and N-linked glycosylation are required for prostatic acid phosphatase catalytic and antinociceptive activity

Secretory human prostatic acid phosphatase (hPAP) is glycosylated at three asparagine residues (N62, N188, N301) and has potent antinociceptive effects when administered to mice. Currently, it is unknown if these N-linked residues are required for hPAP protein stability and activity in vitro or in animal models of chronic pain. Here, we expressed wild-type hPAP and a series of Asn to Gln point mutations in the yeast Pichia pastoris X33 then analyzed protein levels and enzyme activity in cell lysates and in conditioned media. Pichia secreted wild-type recombinant (r)-hPAP into the media (6-7 mg protein/L). This protein was as active as native hPAP in biochemical assays and in mouse models of inflammatory pain and neuropathic pain. In contrast, the N62Q and N188Q single mutants and the N62Q, N188Q double mutant were expressed at lower levels and were less active than wild-type r-hPAP. The purified N62Q, N188Q double mutant protein was also 1.9 fold less active in vivo. The N301Q mutant was not expressed, suggesting a critical role for this residue in protein stability. To explicitly test the importance of secretion, a construct lacking the signal peptide of hPAP was expressed in Pichia and assayed. This "cellular" construct was not expressed at levels detectable by western blotting. Taken together, these data indicate that secretion and post-translational carbohydrate modifications are required for PAP protein stability and catalytic activity. Moreover, our findings indicate that recombinant hPAP can be produced in Pichia--a yeast strain that is used to generate biologics for therapeutic purposes.     

At5g50600 encodes a member of the short-chain dehydrogenase reductase superfamily with 11beta- and 17beta-hydroxysteroid dehydrogenase activities associated with Arabidopsis thaliana seed oil bodies

In a previous work, we presented evidence for the presence of a protein encoded by At5g50600 in oil bodies (OBs) from Arabidopsis thaliana [P. Jolivet, E. Roux, S. D'Andrea, M. Davanture, L. Negroni, M. Zivy, T. Chardot, Protein composition of oil bodies in Arabidopsis thaliana ecotype WS, Plant Physiol. Biochem. 42 (2004) 501-509]. Using specific antibodies and proteomic techniques, we presently confirm the existence of this protein, which is a member of the short-chain steroid dehydrogenase reductase superfamily. We have measured its activity toward various steroids (cholesterol, dehydroepiandrosterone, cortisol, corticosterone, estradiol, estrone) and NAD(P)(H), either within purified OBs or as a purified bacterially expressed chimera. Both enzymatic systems (OBs purified from A. thaliana seeds as well as the chimeric enzyme) exhibited hydroxysteroid dehydrogenase (HSD) activity toward estradiol (17beta-hydroxysteroid) with NAD+ or NADP+, NADP+ being the preferred cofactor. Low levels of activity were observed with cortisol or corticosterone (11beta-hydroxysteroids), but neither cholesterol nor DHEA (3beta-hydroxysteroids) were substrates, whatever the cofactor used. Similar activity profiles were found for both enzyme sources. Purified OBs were found to be also able to catalyze estrone reduction (17beta-ketosteroid reductase activity) with NADPH. The enzyme occurring in A. thaliana OBs can be classified as a NADP+-dependent 11beta-,17beta-hydroxysteroid dehydrogenase/17beta-ketosteroid reductase. This enzyme probably corresponds to AtHSD1, which is encoded by At5g50600. However, its physiological role and substrates still remain to be determined.     

High-level expression of recombinant phospholipase C from Bacillus cereus in Pichia pastoris and its characterization

The phospholipase c (plc) gene from Bacillus cereus was cloned into the pPICZC vector and integrated into the genome of Pichia pastoris. The phospholipase C (PLC) when expressed in P. pastoris was fused to the alpha-factor secretion signal peptide of Saccharomyces cerevisiae and secreted into a culture medium. Recombinant P. pastoris X-33 had a clear PLC band at 28.5 kDa and produced an extracellular PLC with an activity of 678 U mg(-1) protein which was more than a recombinant P. pastoris GS115 (552 U mg(-1) protein) or KM71H (539 U mg(-1) protein). The PLCs were purified using a HiTrap affinity column with a specific activity of 1335 U mg(-1) protein by P. pastoris GS115, 1176 U mg(-1) protein by P. pastoris KM71H and 1522 U mg(-1) protein by P. pastoris X-33. The three recombinant PLCs had high PLC activity in the low pH range of 4-5 and higher thermal stability (e.g. stable at 75 degrees C) than the wild-type PLC from B. cereus . Some organic solvents, surfactants and metal ions, e.g. methanol, acetone, Co(2+) and Mn(2+) etc., also influenced the activity of the recombinant PLCs.     

Purified NS2B/NS3 serine protease of dengue virus type 2 exhibits cofactor NS2B dependence for cleavage of substrates with dibasic amino acids in vitro

Dengue virus type 2 NS3, a multifunctional protein, has a serine protease domain (NS3pro) that requires the conserved hydrophilic domain of NS2B for protease activity in cleavage of the polyprotein precursor at sites following two basic amino acids. In this study, we report the expression of the NS2B-NS3pro precursor in Escherichia coli as a fusion protein with a histidine tag at the N terminus. The precursor was purified from insoluble inclusion bodies by Ni(2+) affinity and gel filtration chromatography under denaturing conditions. The denatured precursor was refolded to yield a purified active protease complex. Biochemical analysis of the protease revealed that its activity toward either a natural substrate, NS4B-NS5 precursor, or the fluorogenic peptide substrates containing two basic residues at P1 and P2, was dependent on the presence of the NS2B domain. The peptide with a highly conserved Gly residue at P3 position was 3-fold more active as a substrate than a Gln residue at this position. The cleavage of a chromogenic substrate with a single Arg residue at P1 was NS2B-independent. These results suggest that heterodimerization of the NS3pro domain with NS2B generates additional specific interactions with the P2 and P3 residues of the substrates.     

Purification and properties of beta-lactamase from Proteus morganii.

The cephalosporin beta-lactamase was purified from a strain of Proteus morganii that showed resistance to beta-lactam antibiotics and produced the enzyme constitutively. The purified enzyme preparation gave a single protein band on polyacrylamide gel electrophoresis and consisted of a single polypeptide of molecular weight 38,000 to 40,000 from gel filtration of Sephadex G-100 and sodium dodecyl sulfate-acrylamide gel electrophoresis, its isoelectric point being pH 7.2 No cysteine residue was found in its amino acid composition. The specific activity was 190 mumol/min per mg of the purified enzyme protein for the hydrolysis of cephaloridine, the optimal pH was about 8.5 and the optimal temperature was 50 degrees C. Antibodies against the purified beta-lactamase inhibited not only the enzyme activity of the purified preparation, but also the enzyme activity of all of the other strains of P. morganii so far tested, regardless of whether the modes of their production were inducible or constitutive. None of the beta-lactamases produced by beta-lactam antibiotic-resistant strains of other species of Proteus was affected at all by the antibodies, thus showing that the purified cephalosporin beta-lactamase was of the species-specific type. The enzymological properties of the preparation have been compared with those of beta-lactamases derived from other gram-negative enteric bacteria.     

Identification in vitro of a post-translational regulatory site in the hinge 1 region of Arabidopsis nitrate reductase.

Nitrate reductase (NR) is rapidly inactivated by phosphorylation of serine residues in response to loss of light or reduction in CO2 levels. To identify sites within NR protein that play a role in this post-translational regulation, a heterologous expression system and an in vitro inactivation assay for Arabidopsis NR were developed. Protein extracts containing NR kinases and inhibitor proteins were prepared from an NR-defective mutant that had lesions in both the NIA1 and NIA2 NR genes of Arabidopsis. Active NR protein was produced in a Pichia pastoris expression system. Incubation of these two preparations resulted in a Mg-ATP-dependent inactivation of NR that was reversed with EDTA. Mutant forms of NR were constructed, produced in P. pastoris, and tested in the in vitro inactivation assay. Six conserved serine residues in the hinge 1 region of NR, which separates the molybdenum cofactor and heme domains, were specifically targeted for mutagenesis because they are located in a potential regulatory region identified as a target for NR kinases in spinach. A change in Ser-534 to aspartate was found to block NR inactivation; changes in the other five serines had no effect. The aspartate that replaced Ser-534 did not appear to mimic a phosphorylated serine but simply prevented the NR from being inactivated. These results identify Ser-534, located in the hinge 1 of NR and conserved among higher plants NRs, as an essential site for post-translational regulation in vitro.