HO;C'*'^'^X enzyme HO;C**‘^ '^ Y
^
i.
0-succinylhomoserine(thiol)-lyase X=-02CCH2CH2C02H, Y = -cysteine
ii. Oacetylhomosehne(thiol)-lyase X = -O2CCH3, Y = methanethiol
Schem e 1.17b: Some other types of reaction catalysed by PLP dependent enzymes.
The a-Fam ily of PLP Dependent Enzymes.
The a-family has, by far, the most members of all the PLP dependent groups (Table 1.18), including the aminotransferases and the group II decarboxylases. Many of these enzymes have 3D structures available for comparison, and the structural similarities can be seen clearly between different types of enzyme.
Most aminotransferases (excluding branched chain and D-amino acid)®®>^® Group II amino acid decarboxylases
Tryptophanase Tyrosine phenol-lyase^^ Glycine hydroxymethyltransferase
1 -Aminocyclopropane-1 -carboxylate synthase^^ 2-Amino-6-caprolactam racemase
Glutamate-1 -semialdehyde 2,1 -aminomutase Isopenicillin N-epimerase 2,2-dialkyl decarboxylase^® 4-Amino-4-deoxychorismate synthase^"^-^® G lycine-C-acetyltransferase 5-Aminolevulinate synthase 8-Amino-7-oxononanoate synthase
The gene product of cobC (cobalamin synthesis) The gene product of nifS (nitrogen fixation)
The gene product of malY (abolishes induction of the maltose system)
In the a-family the PLP binding lysine residue occurs between residues 209 and 256 of the various members. The most intensely studied members of the a-family are the aminotransferases (or transaminases), which have already been discussed (page 6 and Scheme 1.17a), as have examples of the decarboxylases (Scheme 1,17a), Serine hydroxymethyltransferase catalyses the decarboxylation of aminomalonate as shown in Scheme 1.17a. Tryptophanase and tyrosine phenol- lyase catalyse reactions of type a) (Scheme 1.17b).
The (3-Family of PLP Dependent Enzymes.
The p-family has seven members, and includes threonine synthase (Table 1.19). Parsot first postulated the evolutionary connection of these enzymes in 1967.^®»^^ These suggestions were later upheld by Bork and Rohde.^® If these enzymes are related in an evolutionary context, then they may be distantly related to an archaic enzyme with very broad specificity.
(2S)-Serine dehydratase (2R)-Serine dehydratase Threonine dehydratase
Threonine synthase Tryptophan dehydratase Tryptophan synthase (p-subunit) Cysteine synthase (isoenzyme A and B)
The p-family has the PLP binding lysine lying between amino acid residues 41 and 118. This is clearly very different from the a-family, and may reflect the alternative PLP arrangement needed for the enzymes to carry out reactions at the C«-centre rather than at the CP-atom. The serine dehydratases, threonine dehydratase and tryptophan dehydratase catalyse reactions of type a (Scheme 1.17b). Tryptophan synthase and cysteine synthase catalyse reactions of type b, and threonine synthase catalyses a reaction of type c.
The y-Fam ily of PLP Dependent Enzymes.
This is the smallest "family" of enzymes with only four members (Table 1.20). Interestingly Bork and Rohde have shown significant sequence similarities between threonine synthase and O-acetylserine sulfhydrolase.^® This may suggest that the p- and y- families are more closely related than Christen’s work would suggest on first examination. On the other hand, the fact that threonine synthase also catalyses a change at the CY-atom of its substrate may account for this similarity.
0-Succinylhomoserine(thiol)-Iyase 0-Acetylhomoserine(thiol)-Iyase
Cystathionine p-lyase Cystathionine y-lyase
In the y-family PLP-Lys occurs in the same sequence segment as in the a-family, thus hinting at a possible evolutionary relationship between the two. This could be further investigated by comparison of the 3D structures of a - and y- family members, when the structure of a y-family member becomes available. 0-succinylhomoserine(thiol)-lyase and 0-acetylhomoserine(thiol)-lyase catalyse similar reactions of type e (Scheme 1.17b). Cystathionine p-synthase catalyses a reaction of type a (acting at the p-carbon as the name suggests), and the closely related cystathionine y-synthase catalyses the similar reaction d.
Exceptions to the "Family Rule".
As already mentioned, a few PLP dependent enzymes do not belong to the family that would be suggested by their regioselectivity. Tryptophanase, tyrosine phenol- lyase and 4-amino-4-deoxychorismate synthase are all members of the a-family by sequence, yet catalyse p-elimination reactions; whereas 1 -aminocyclopropane-1 - carboxylate synthase, another a-family enzyme, catalyses an a,y-replacement. Similarly, threonine synthase which as we have seen assigned to the p-family, catalyses a p,y-replacement reaction; and cystathionine p-lyase (a y-family member) catalyses a p-elimination.
With increased access to sequence searching and alignment software, the evolutionary history of threonine synthase and other PLP dependent enzymes is gaining more attention. Using up to date computer technology it may be possible to reliably fit related enzyme sequences to already solved structures to increase our understanding of these enzymes.
1.4 Introduction to Threonine Synthase.
Pyridoxal 5'-phosphaîe enzymes had been known about for over two decades (see section 1.0) when, in 1960, Flavin and Slaughter reported a new and quite different pyridoxal 5'-phosphate dependent enzyme. In their words, the enzyme "...which will be called threonine synthetase, catalysed an elimination of orthophosphate coupled to isomérisation from a- to p- hydroxy compound, to yield threonine from O-phosphohom oserine.".^® Now more commonly referred to as threonine synthase (EC 4.2.99.2), the enzyme is known to stereospecifically catalyse the conversion of (2S)-0-phosphohom oserine (43) to (2S)-threonine (44) and inorganic phosphate in the final step of threonine biosynthesis in plants (Scheme
1
.
21).2' ' O H
HOpC HOpC