NORMAL GENOMIC

• Seah SY, Britton KL, Rice DW, Asano Y, Engel PC
• Kinetic analysis of phenylalanine dehydrogenase mutants designed foraliphatic amino acid dehydrogenase activity with guidance fromhomology-based modelling.
• Eur J Biochem. 2003; 270: 4628-34
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• Through comparison with the high-resolution structure of Clostridiumsymbiosum glutamate dehydrogenase, the different substrate specificitiesof the homologous enzymes phenylalanine dehydrogenase and leucinedehydrogenase were attributed to two residues, glycine 124 and leucine307, in Bacillus sphaericus phenylalanine dehydrogenase, which arereplaced with alanine and valine in leucine dehydrogenases. As predicted,making these substitutions in phenylalanine dehydrogenase decreased thespecific activity towards aromatic substrates and enhanced the activitytowards some aliphatic amino acids in standard assays with fixedconcentrations of both substrates. This study did not, however,distinguish effects on affinity from those on maximum catalytic rate. Afuller kinetic characterization of the single- and double-mutant enzymesnow reveals that the extent of the shift in specificity was underestimatedin the earlier study. The maximum catalytic rates for aromatic substratesare reduced for all the mutants, but, in addition, the apparent Km valuesare higher for the single-mutant G124A and double-mutant G124A/L307Vcompared with the wild-type enzyme. Conversely, specificity constants(kcat/Km) for the nonpolar aliphatic amino acids and the corresponding2-oxoacids for the mutants are all markedly higher than for the wild type,with up to a 40-fold increase for l-norvaline and a 100-fold increase forits 2-oxoacid in the double mutant. In some cases a favourable change inKm was found to outweigh a smaller negative change in kcat. These resultsemphasize the risk of misjudging the outcome of protein engineeringexperiments through too superficial an analysis. Overall, however, thesuccess of the predictions from molecular modelling indicates theusefulness of this strategy for engineering new specificities, even inadvance of more detailed 3D structural information.

• Seah SY, Britton KL, Rice DW, Asano Y, Engel PC
• Single amino acid substitution in Bacillus sphaericus phenylalaninedehydrogenase dramatically increases its discrimination betweenphenylalanine and tyrosine substrates.
• Biochemistry. 2002; 41: 11390-7
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• Homology-based modeling of phenylalanine dehydrogenases (PheDHs) fromvarious sources, using the structures of homologous enzymes Clostridiumsymbiosum glutamate dehydrogenase and Bacillus sphaericus leucinedehydrogenase as a guide, revealed that an asparagine residue at position145 of B. sphaericus PheDH was replaced by valine or alanine in PheDHsfrom other sources. This difference was proposed to be the basis for thepoor discrimination by the B. sphaericus enzyme between the substratesL-phenylalanine and L-tyrosine. Residue 145 of this enzyme was altered, bysite-specific mutagenesis, to hydrophobic residues alanine, valine,leucine, and isoleucine, respectively. The resultant mutants showed a highdiscrimination, above 50-fold, between L-phenylalanine and L-tyrosine.This higher specificity toward L-phenylalanine was due to K(m) values forL-phenylalanine lowered more than 20-fold compared to the values forL-tyrosine. The greater specificity for L-phenylalanine in the wild-typeBacillus badius enzyme, which has a valine residue in the correspondingposition, was also found to be largely due to a lower K(m) for thissubstrate. Activities were also measured with a range of six amino acidswith aliphatic, nonpolar side chains, and with the corresponding oxoacids,and in all cases the specificity constants for these substrates wereincreased in the mutant enzymes. As with phenylalanine, these increasesare mainly attributable to large decreases in K(m) values.

• Ruzheinikov SN et al.
• Glycerol dehydrogenase. structure, specificity, and mechanism of a familyIII polyol dehydrogenase.
• Structure. 2001; 9: 789-802
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• BACKGROUND: Bacillus stearothermophilus glycerol dehydrogenase (GlyDH)(glycerol:NAD(+) 2-oxidoreductase, EC 1.1.1.6) catalyzes the oxidation ofglycerol to dihydroxyacetone (1,3-dihydroxypropanone) with concomitantreduction of NAD(+) to NADH. Analysis of the sequence of this enzymeindicates that it is a member of the so-called iron-containing alcoholdehydrogenase family. Despite this sequence similarity, GlyDH shows astrict dependence on zinc for activity. On the basis of this, we proposeto rename this group the family III metal-dependent polyol dehydrogenases.To date, no structural data have been reported for any enzyme in thisgroup. RESULTS: The crystal structure of B. stearothermophilus glyceroldehydrogenase has been determined at 1.7 A resolution to providestructural insights into the mechanistic features of this family. Theenzyme has 370 amino acid residues, has a molecular mass of 39.5 kDa, andis a homooctamer in solution. CONCLUSIONS: Analysis of the crystalstructures of the free enzyme and of the binary complexes with NAD(+) andglycerol show that the active site of GlyDH lies in the cleft between theenzyme's two domains, with the catalytic zinc ion playing a role instabilizing an alkoxide intermediate. In addition, the specificity of thisenzyme for a range of diols can be understood, as both hydroxyls of theglycerol form ligands to the enzyme-bound Zn(2+) ion at the active site.The structure further reveals a previously unsuspected similarity todehydroquinate synthase, an enzyme whose more complex chemistry shares acommon chemical step with that catalyzed by glycerol dehydrogenase,providing a striking example of divergent evolution. Finally, thestructure suggests that the NAD(+) binding domain of GlyDH may be relatedto that of the classical Rossmann fold by switching the sequence order ofthe two mononucleotide binding folds that make up this domain.

• Stillman TJ et al.
• Insights into the mechanism of domain closure and substrate specificity ofglutamate dehydrogenase from Clostridium symbiosum.
• J Mol Biol. 1999; 285: 875-85
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• Comparisons of the structures of glutamate dehydrogenase (GluDH) andleucine dehydrogenase (LeuDH) have suggested that two substitutions, deepwithin the amino acid binding pockets of these homologous enzymes, fromhydrophilic residues to hydrophobic ones are critical components of theirdifferential substrate specificity. When one of these residues, K89, whichhydrogen-bonds to the gamma-carboxyl group of the substrate l-glutamate inGluDH, was altered by site-directed mutagenesis to a leucine residue, themutant enzyme showed increased substrate activity for methionine andnorleucine but negligible activity with either glutamate or leucine. Inorder to understand the molecular basis of this shift in specificity wehave determined the crystal structure of the K89L mutant of GluDH fromClostridium symbiosum. Analysis of the structure suggests that furthersubtle differences in the binding pocket prevent the mutant from using abranched hydrophobic substrate but permit the straight-chain amino acidsto be used as substrates.The three-dimensional crystal structure of theGluDH from C. symbiosum has been previously determined in two distinctforms in the presence and absence of its substrate glutamate. A comparisonof these two structures has revealed that the enzyme can adopt differentconformations by flexing about the cleft between its two domains,providing a motion which is critical for orienting the partners involvedin the hydride transfer reaction. It has previously been proposed thatthis conformational change is triggered by substrate binding. However,analysis of the K89L mutant shows that it adopts an almost identicalconformation with that of the wild-type enzyme in the presence ofsubstrate. Comparison of the mutant structure with both the wild-type openand closed forms has enabled us to separate conformational changesassociated with substrate binding and domain motion and suggests that thedomain closure may well be a property of the wild-type enzyme even in theabsence of substrate.

• Baker PJ, Sawa Y, Shibata H, Sedelnikova SE, Rice DW
• Analysis of the structure and substrate binding of Phormidium lapideumalanine dehydrogenase.
• Nat Struct Biol. 1998; 5: 561-7
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• The structure of the hexameric L-alanine dehydrogenase from Phormidiumlapideum reveals that the subunit is constructed from two domains, eachhaving the common dinucleotide binding fold. Despite there being nosequence similarity, the fold of alanine dehydrogenase is closely relatedto that of the family of D-2-hydroxyacid dehydrogenases, with a similarlocation of the active site, suggesting that these enzymes are related bydivergent evolution. L-alanine dehydrogenase and the 2-hydroxyaciddehydrogenases also use equivalent functional groups to promote substraterecognition and catalysis. However, they are arranged differently on theenzyme surface, which has the effect of directing opposite faces of theketo acid to the dinucleotide in each case, forcing a change in absoluteconfiguration of the product.

• Baker PJ et al.
• Determinants of substrate specificity in the superfamily of amino aciddehydrogenases.
• Biochemistry. 1997; 36: 16109-15
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• The subunit of the enzyme glutamate dehydrogenase comprises two domainsseparated by a cleft harboring the active site. One domain is responsiblefor dinucleotide binding and the other carries the majority of residueswhich bind the substrate. During the catalytic cycle a large movementbetween the two domains occurs, closing the cleft and bringing the C4 ofthe nicotinamide ring and the Calpha of the substrate into the correctpositioning for hydride transfer. In the active site, two residues, K89and S380, make interactions with the gamma-carboxyl group of the glutamatesubstrate. In leucine dehydrogenase, an enzyme belonging to the samesuperfamily, the equivalent residues are L40 and V294, which create a morehydrophobic specificity pocket and provide an explanation for theirdifferential substrate specificity. In an attempt to change the substratespecificity of glutamate dehydrogenase toward that of leucinedehydrogenase, a double mutant, K89L,S380V, of glutamate dehydrogenase hasbeen constructed. Far from having a high specificity for leucine, thismutant appears to be devoid of any catalytic activity over a wide range ofsubstrates tested. Determination of the three-dimensional structure of themutant enzyme has shown that the loss of function is related to adisordering of residues linking the enzyme's two domains, probably arisingfrom a steric clash between the valine side chain, introduced at position380 in the mutant, and a conserved threonine residue, T193. In leucinedehydrogenase the steric clash between the equivalent valine and threonineside chains (V294, T134) does not occur owing to shifts of the main chainto which these side chains are attached. Thus, the differential substratespecificity seen in the amino acid dehydrogenase superfamily arises fromboth the introduction of simple point mutations and the fine tuning of theactive site pocket defined by small but significant main chainrearrangements.

• Turnbull AP, Baker PJ, Rice DW
• Analysis of the quaternary structure, substrate specificity, and catalyticmechanism of valine dehydrogenase.
• J Biol Chem. 1997; 272: 25105-11
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• The solution of the three-dimensional structure of Bacillus sphaericusleucine dehydrogenase has enabled us to undertake a homology-basedmodeling exercise on the sequence differences between the families ofleucine (LeuDH) and valine (ValDH) dehydrogenases. This analysis indicatesthat the secondary structure elements in the core of the two domains of asingle subunit of these enzymes are conserved, as are residues directlyimplicated in the recognition of the nucleotide cofactor and in catalysis.Comparison of the sequences indicates that the residues in the pocketaccommodating the side chain of the amino acid substrate are conservedbetween these two enzymes, suggesting that the small differences inspecificity arise from minor changes in molecular structure, possiblyassociated with shifts of the main chain rather than mutation of residuesin the pocket itself. While B. sphaericus LeuDH is an octamer, bothStreptomyces cinnamonensis and Streptomyces coelicolor ValDHs are dimers.The differences in quaternary structure can be understood in terms of thedeletion in the latter of a C-terminal loop, which forms importantinteractions around the four-fold axis in LeuDH.

• Kataoka K et al.
• Construction and characterization of chimeric enzyme consisting of anamino-terminal domain of phenylalanine dehydrogenase and acarboxy-terminal domain of leucine dehydrogenase.
• J Biochem. 1994; 116: 931-6
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• Phenylalanine dehydrogenase of Thermoactinomyces intermedius actspreferentially on L-phenylalanine and L-tyrosine, whereas leucinedehydrogenase of Bacillus stearothermophilus acts almost exclusively onL-leucine and some other branched-chain L-amino acids. The two enzymesshare a sequence similarity (47%). Aiming at elucidation of the mechanismof substrate recognition by the two amino acid dehydrogenases, we havegenetically constructed a chimeric enzyme consisting of an N-terminaldomain of phenylalanine dehydrogenase containing the substrate-bindingregion and a C-terminal domain of leucine dehydrogenase containing theNAD(+)-binding region. The chimeric enzyme purified to homogeneity actedon phenylalanine with a specific activity of 6% of that of the parentalphenylalanine dehydrogenase and showed a broad substrate specificity inthe oxidative deamination, like phenylalanine dehydrogenase. However, itacted much more effectively than phenylalanine dehydrogenase on isoleucineand valine. Its Km values for L-phenylalanine and L-leucine were similarto those of phenylalanine dehydrogenase. The substrate specificity of thechimeric enzyme in the reductive amination was an admixture of those ofthe two parent enzymes. These results suggest that the two domains ofphenylalanine dehydrogenase and leucine dehydrogenase probably can foldindependently. Accordingly, their chimera forms a new active enzyme whichconsists of their N- and C-terminal domains containing the substrate- andcoenzyme-binding regions, respectively. However, the two domains ofchimeric enzyme interact and communicate with each other to form a newactive site and consistently show the new substrate specificity.

• Turnbull AP et al.
• Crystallization and quaternary structure analysis of the NAD(+)-dependentleucine dehydrogenase from Bacillus sphaericus.
• J Mol Biol. 1994; 236: 663-5
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• The NAD(+)-dependent leucine dehydrogenase from Bacillus sphaericus hasbeen crystallized by the hanging drop method of vapour diffusion, usingammonium sulphate as the precipitant. The crystals belong to thetetragonal system and are in space group I4, with unit cell dimensions ofa = b = 138.4 A and c = 121.8 A. Considerations of the values of Vm, thespace group symmetry and an analysis of a self-rotation functioncalculated on a preliminary data set collected to 3 A resolution show thatthe asymmetric unit contains a dimer with the twofold axis perpendicularto the crystallographic four fold, indicating that the quaternarystructure of this enzyme is octameric. Leucine dehydrogenase belongs to asuperfamily of amino acid dehydrogenases which display considerabledifferences in amino acid specificity and elucidation of itsthree-dimensional structure should enable the molecular basis of thisdifferential specificity to be examined in detail.

• Britton KL, Baker PJ, Engel PC, Rice DW, Stillman TJ
• Evolution of substrate diversity in the superfamily of amino aciddehydrogenases. Prospects for rational chiral synthesis.
• J Mol Biol. 1993; 234: 938-45
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• We have analysed the sequence homology between glutamate, leucine andphenylalanine dehydrogenases in the light of the solution of the structureof the glutamate dehydrogenase from Clostridium symbiosum. This analysisindicates that the elements of secondary structure comprising the core ofthe two domains in glutamate dehydrogenase are conserved in the other twoenzymes. There is a striking conservation of the residues responsible forthe recognition of the nicotinamide ring of the nucleotide cofactor andthe backbone of the amino acid substrates. Furthermore, residues involvedin a major conformational rearrangement on amino acid binding arepreserved, as are those implicated in the catalytic chemistry. Incontrast, the pattern of insertions/deletions between these enzymes isconsistent with possible differences in quaternary structure. Differentialsubstrate specificity between these enzymes is achieved by criticalsubstitutions at the base of the binding pocket, which accommodates theside-chain of the amino acid substrate. This provides insights into themutations necessary to produce new catalysts for the chiral synthesis ofnovel amino acids.