Pre-Loading of Chlorophyll Synthase with Tetraprenyl Diphosphate Is an Obligatory Step in Chlorophyll Biosynthesis

Abstract

The reaction of recombinant chlorophyll synthase from Avena sativa, expressed in Escherichia coli, was investigated. To verify the identity of the recombinant and native enzymes, reaction rates were determined for both enzyme preparations with several chlorophyllide analogs. The rates of esterification of these modified substrates ranged from 0 to 100% of the rate with the natural substrate, and were nearly identical for both enzyme preparations. The LineweaverBurk plot for variation of both chlorophyllide a and phytyl diphosphate concentration showed parallel lines, indicative of a 'ping-pong' mechanism. Pre-incubation with phytyl diphosphate exhibited an initial rapid reaction phase, which did not occur after preincubation with chlorophyllide. We conclude that the tetraprenyl diphosphate must bind to the enzyme as the first substrate and esterification occurs when this pre-loaded enzyme meets the second substrate, chlorophyllide. Approximately 10 -17% of the recombinant enzyme were pre-loaded with phytyl diphosphate under the experimental conditions. The rapid reaction phase is also observed for the chlorophyll synthase reaction in etiolated barley leaves in addition to the well-known slow phase. This indicates that pre-loading of the enzyme with tetraprenyl diphosphate is also the basis for the reaction in vivo.

Competitive Inhibitions of the Chlorophyll Synthase of Synechocystis sp. Strain PCC 6803 by Bacteriochlorophyllide a and the Bacteriochlorophyll Synthase of Rhodobacter sphaeroides by Chlorophyllide a

“…No differences in rates between the two were observed thereafter. Thus, enzyme phytylation is a rate-limiting step, as observed with chlorophyll synthase from A. sativa (27). The result also reflects that PPP may be the first substrate, since the accelerated rate would be expected only if the first reaction were rate limiting and completed during preincubation.…”

“…Since the enzyme was examined in terms of whether its esterification is affected by the presence of Bchlide a, the reaction was performed in the presence of a sufficient level (200 M) of geranylgeranyl pyrophosphate (GGPP). However, GGPP, once mixed with E. coli lysate, is readily metabolized to lower its effective concentration (27), so the velocity (V 0 ) was measured during the initial 15 min, in which the reaction rate falls within a linear range (data not shown). The K m value (0.1 mM) of chlorophyll synthase for Chlide a (Table 1) apparently increased in the presence of Bchlide a (140 M) (Fig.…”

“…The results are diagnostic of competitive inhibition with an inhibition constant (K I ) for Bchlide a binding of 0.30 mM (Table 1). Phytyl pyrophosphate (PPP), which is also a natural substrate for chlorophyll synthase, is not metabolized rapidly in E. coli lysate (27). Accordingly, PPP was employed as a substrate instead of GGPP.…”

“…Whether chlorophyll synthase of Synechocystis sp. PCC 6803 follows a ping-pong reaction as observed with chlorophyll synthase from the oat plant Avena sativa (27) was investigated. The double-reciprocal (Lineweaver-Burk) plots of the chlorophyll synthase reaction were determined either with various concentrations of Chlide a and four fixed concentrations of PPP (Fig.…”

“…The C 20 moiety from geranylgeranyl pyrophosphate (GGPP) can be directly esterified to ring D of Chlide a by chlorophyll synthase (ChlG) to yield geranylgeranylated Chl a (Chl a gg ), which is subsequently reduced (at positions 6, 10, and 14 of GG) by chlorophyll reductase (ChlP) to yield phytylated Chl a (Chl a p , but it is usually abbreviated as Chl a) (2,7). The chlorophyll synthase of Avena sativa has a broad substrate specificity for C 20 , and it may accept either GGPP or phytyl pyrophosphate (PPP) as the first substrate in its ping-pong-type reaction (27). Either a geranylgeranylated or phytylated enzyme esterifies the second substrate Chlide a, yielding Chl a gg or Chl a, respectively (5,24).…”